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Protocols

Protein expression, periplasmic extraction and IMAC purification

Author: Nyytti Koskinen

The sources this protocol is based on:



You can find information about the protocol’s success and modifications made to subsequent protocols here.



Preculture: Day 1

  1. Start heating up the shaker to 37 °C.
  2. Pick a fresh colony of BL21(DE3)pLysS with your expression vector, and inoculate it in 4 mL of LB supplemented with relevant antibiotics. We used 50 µg/mL kanamycin and 100 µg/mL ampicillin as these are the selection markers for our vectors.
  3. Grow the culture at 37 °C at 250 rpm overnight.


Expression: Day 2

  1. Add 1.5 mL of preculture to 100 mL of fresh LB supplemented with relevant antibiotics in a 500 mL flask. We used 50 µg/mL kanamycin and 100 µg/mL ampicillin as these are the selection markers for our vectors.
  2. Grow the culture at 37 °C with 250 rpm until an OD600 =0.5-0.6
  3. Induce the expression by adding IPTG to a final concentration of 1 mM.
  4. Let the culture grow at 18 °C with 180 rpm shaking for 20 hours.


Periplasmic extraction: Day 3

  1. Spin the culture down at 8,000 x g 4 °C 20 min and discard the supernatant.
  2. Carefully resuspend in 3 mL of TSE buffer (200 mM Tris-HCl pH 8, 500 mM sucrose, 1 mM EDTA) per gram of cells.
  3. Incubate the suspension at room temperature for 10 min.
  4. Cold-shock the cells by adding 3 mL of ice-cold MQ water per gram of cells.
  5. Incubate the suspension on ice for 10 min.
  6. Spin down the cells at 4,969 x g 4 °C 35 min and collect the supernatant.
  7. Do Bradford assay to the periplasmic extracts.
  8. Keep the extraction on ice when working with it and at 4 °C for storage.


Batch spin IMAC purification: Day 3

All steps are done using cold buffer solutions and centrifuging at +4 °C.

Buffer A: 20 mM sodium phosphate pH 7, 100 mM NaCl + 5 mM imidazole + 10 % glycerol

Buffer B: 20 mM sodium phosphate pH 7, 100 mM NaCl + 250 mM imidazole+ 10 % glycerol

  1. Pretreatment of the Co-resin.
    • Take about 0.5 mL of unused slurry (in 20 % EtOH) to a 15 mL falcon tube.
    • 5 mL MQ water is added and the suspension is centrifuged 10 min 700 x g.
    • Remove the storage buffer on top with a pipette.
    • The pellet is resuspended in 10 mL of Buffer A (5 mM imidazole) and the resin is pelleted again 10 min 700 x g 4 °C.
    • Remove the excess Buffer A on top with a pipette.
    • The pellet is resuspended in 10 mL of Buffer A (5 mM imidazole) again and the resin is pelleted again 10 min 700 x g 4 °C.
    • Remove the excess Buffer A on top with a pipette. Keep pelleted resin on ice.
  2. Binding of the His-tagged protein.
    • Add 5 mL of buffer A to protein extractions.
    • Add protein extraction to the reaction tube.
    • The His-tagged protein is allowed to bind to the resin for 30-45 min with gentle mixing at 4 °C.
    • Centrifuge for 15 min at 700 x g 4 °C.
    • Remove the excess protein extraction with a pipette. Take a sample for measurements with Bradford assay and SDS-PAGE.
    • A second centrifugation of the supernatant may be necessary for effective pelleting of the resin.
  3. Washing out unbound protein.
    • The resin pellet is resuspended in a total volume about 10 mL of cold Buffer A and incubated with gentle shaking for about 10 min before centrifugation.
    • Centrifuge 10 min 700 x g 4 °C.
    • Remove the buffer on top with a pipette.
    • Repeat four times.
  4. Elution.
    • Add 2 mL of Buffer B.
    • Incubate 10 min 4 °C with shaking.
    • Centrifuge 10 min at 700 x g 4 °C.
    • Move the supernatant on top with a pipette to a centrifuge tube. This supernatant contains the protein!
    • Repeat four times, so five times in total.
    • Do Bradford assay for each 5 fractions.

Author: Nyytti Koskinen

This protocol is based on:



This protocol has been modified from the first version of the protocol. Things such as culture conditions and buffer pH values have been modified. This protocol also includes lysis. You can find information about the protocol’s success and modifications made to subsequent protocols here.



Day 1: preculture

Start this protocol earliest at 15.00.

Note: Work in a laminar flow hood.

  1. Set up the laminar flow hood with inoculation loops and plates. Start heating up the shaker to 37 °C.
  2. Go get glycerol preps for strains you want to use for protein expression from -80 °C one at a time. Do not let the glycerol prep thaw. Keep glycerol prep on ice and take it back to the freezer as soon as possible. Remember to get the control strain (does not have expression plasmid inside) glycerol prep as well.
  3. Scrape of some frozen bacteria from the glycerol prep with a sterile inoculation loop in a laminar flow hood.
  4. Streak bacteria onto the LA plate with relevant antibiotics. The control strain may be plated to a LA plate without any antibiotics. Remember to take a new sterile inoculation loop for each streak. Make two plates per prep. The partner will use this time to take the prep back to the freezer and get the next one. Make one control plate by streaking with a sterile inoculation loop without adding any bacteria from glycerol preps.
  5. Incubate overnight at 37 °C. Take the plates to the incubator earliest at 16.


Day 2: Overnight liquid culture

Note: Work in a laminar flow hood.

  1. At 9: get the plates from the incubator. Put successful plates (single colonies) to the fridge and take unsuccessful ones to the GMO plate trash. The control plate is successful if nothing grows on it.
  2. Earliest at 21: Set the incubator at 37 °C. Take the plates from the fridge and take them to the laminar room.
  3. Pipette 4 mL of sterilized LB medium with 0.5 % D-glucose into 14 mL snap cap tubes. Add relevant antibiotics to the media. Remember to mix the antibiotic before pipetting. Do not add antibiotics for the control strain tube! Make 1 preculture per strain.
  4. Add a colony to the tube in a sterile manner using an inoculation loop to the tube.
  5. Grow cells for 18 hours at 37 °C 250 rpm. Remember to leave the tube tilted and the cap ajar. Put the plates in the fridge. Move IPTG from the freezer to the fridge for thawing.


Day 3: Expression

Start the protocol after 18 hours have passed since putting the overnight culture to the incubator so at about 16.

  1. Write the name of the protein on the 500 mL flasks. Add relevant antibiotics to the flasks. Remember to mix the antibiotic before pipetting. Do not add antibiotics for the control strain! You should have 1 x 500 mL flask per strain.
  2. Blank the spectrophotometer with 1 mL of media.
  3. Add 1 mL of preculture to expression cultures. Put the rest of the preculture to the liquid GMO waste.
  4. Grow expression cultures at 37 °C with 250 rpm shaking until OD600=0.5-0.6. Measure OD600 in a sterile manner. Each measurement required 1 ml of media. You can reuse the cuvette, if you wash with MQ water between measurements. Pour the media after measuring to liquid GMO waste. Remember that E. coli doubling time is about 20 minutes.
  5. Induce the expression by adding IPTG to a final concentration of 0.2 mM. For 100 mL of culture you need 0.2 mL of IPTG. While inducing, the partner will set the incubator at 26 °C. After inducing, take the IPTG back to the freezer.
  6. Grow the cultures at 26 °C with 250 rpm for 12 hours.


Day 4: periplasmic extraction

From this point, we are no longer working in a sterile manner.

  • The partner should use the time that goes into periplasmic extraction to regenerate HisPur Co-NTA resin if necessary. Resin recharging is done according to this protocol.
  1. Get the TSE buffer (200 mM Tris-HCl pH 7.5, 500 mM sucrose, 1 mM EDTA) from the fridge to room temperature.
  2. Cool the centrifuge to 4 °C.
  3. Get the expression cultures from the shaker after 12 hours have passed so at about 8.
  4. Move the cultures to 50 mL tubes by pouring. Weight the tubes to find out the centrifuge pairs. The weights should not differ more than 0.1 g!
  5. Remember to add the centrifuge adapters! Spin the culture down at 8,000 x g, 4 °C for 20 min.
  6. Discard the supernatant by pouring into liquid GMO waste and tap on paper to get rid of as much of the liquid as possible. The paper goes to solid GMO waste.
  7. Weight the cells approximately.
  8. Carefully resuspend the pellet in 3 mL TSE (200 mM Tris-HCl pH 7.5; 500 mM sucrose, 1 mM EDTA) per gram of cells. Combine the same protein cultures.
  9. Incubate the suspension at room temperature for 10 min.
  10. Cold-shock the cells by adding 3 mL of ice-cold MQ water per gram of cells.
  11. Incubate the suspension on ice for 10 minutes.
  12. Centrifuge the cells at 8,000 x g, 4 °C for 20 minutes. Collect the supernatants to 15 mL tubes. Keep on the pellets and supernatants on ice!
  13. Do Bradford assay for the periplasmic extractions on a 96-well plate. Add the Bradford reagent in a fume hood.
  14. Measure total protein concentration with Tecan Infinite plate reader.
  15. Take a 500 µL sample of every extraction to 2 mL tube and add the same volume of 50 % glycerol to the tube in a sterile manner. Put the eppendorf tubes in the freezer. Keep the rest of the extractions in the fridge before starting with the purification.


Day 4: Lysis by sonication

Keep the samples on ice at all times.

  1. Resuspend the pellets in 5 mL of PBS. Keep the cells on ice during the protocol.
  2. Add 15 ml of 1 x PBS pH 7.42 and mix.
  3. Use the sonicator to break the cells with a 4 x 30 s cycle. Keep the cells on ice at all times. The tip should be well-submerged in the suspension, but not touch the walls of the tube. Between each cycle, let the samples incubate on ice for 1 min to allow them to cool. Instructions vary between sonicators.
  4. After lysis, centrifuge 8,000 x g, 4 °C for 20 min.
  5. Collect the supernatant and do Bradford assay.
  6. Add 50 % glycerol as much as the volume of the supernatant. Put the tubes in the freezer.


Day 4: IMAC purification

Keep the buffers on ice.

  1. Pretreatment of the co-resin.
    • Take the used slurries for the correct proteins from the fridge.
    • Add 5 mL MQ water and centrifuge 5 min 700 x g.
    • Remove the storage buffer on top with a pipette.
    • Resuspend the pellet in 10 mL of Buffer A (20 mM sodium phosphate pH 7.5; 100 mM NaCl + 5 mM imidazole + 10 % glycerol) and pellet the resin again by centrifuging 5 min 700 x g.
    • Remove the excess Buffer A on top with a pipette.
    • Resuspend the pellet in 10 mL of Buffer A (5 mM imidazole) again and pellet the resin again 5 min 700 x g.
    • Remove the excess Buffer A on top with a pipette. Keep pelleted resin on ice.
  2. Binding of the His-tagged protein.
    • Add 5 mL of buffer A to periplasmic extractions.
    • Add protein extraction (the ones without added glycerol) to the reaction tube.
    • The His-tagged protein is allowed to bind to the resin for 30-45 min with gentle mixing at 4 °C degrees.
    • Centrifuge for 15 min at 700 x g 4 °C degrees.
    • Remove the excess protein extraction with a pipette. Take a 500 µL sample from every flow-through to a 2 mL centrifuge tube. Do Bradford assay. After that, add 500 µL of 50 % glycerol in a sterile manner. Put them in -20 °C.
    • A second centrifugation of the supernatant may be necessary for effective pelleting of the resin.
  3. Washing out unbound protein.
    • The resin pellet is resuspended in a total volume about 10 mL of cold Buffer A and incubated with gentle shaking for about 10 min before centrifuging.
    • Centrifuge 5 min 700 x g 4 °C degrees.
    • Remove the buffer on top by pouring and tapping gently.
    • Repeat the wash process three times total.
  4. Elution.
    • Add 2 mL of Buffer B (20 mM sodium phosphate pH 7.5; 100 mM NaCl + 250 mM imidazole + 10 % glycerol).
    • Incubate 10 min 4 °C degrees with shaking.
    • Centrifuge 5 min at 700 x g 4 °C degrees.
    • Move the supernatant on top with a pipette to an 1,5 mL centrifuge tube. This supernatant contains the protein!
    • Repeat three times total.
    • Keep fractions that contain protein (based on Bradford assay) and add the same volume of 50 % glycerol in a sterile manner. Put it in -20 °C.
    • Analysis of eluate fractions.Do Bradford assay for the fractions and on a 96-well plate. Add the Bradford in a fume hood.
    • Measure concentrations with the plate reader.

At the end of the day, Bradford assay should have been performed on all fractions, lysates, flow-through and extractions.



Day 5: SDS-PAGE

Refer to the SDS-PAGE protocol for more details.

  1. Remove the comb and tape from the gels and assemble the electrophoresis cell.
  2. Fill the inner and outer buffer chambers with the running buffer.
  3. Prepare the samples in the 2 mL centrifuge tube by adding 15 µL protein sample and 5 µL 4 x SLB.
  4. Heat the samples at 90-100 °C for 5 min in the heat block with water in the wells. Load 10 µL of the sample on the gel. Remember to load 2 ladders (3 µL each) as well.
  5. Connect the electrophoresis cell to the power supply and perform electrophoresis. Run conditions: 200 V
  6. After the run is complete, turn the power supply off and disconnect the electrical leads. Pop open the gel cassettes and remove the gel by floating it off the plate into water.
  7. Wash the gel in MQ for 10 minutes three times.
  8. Add PageBlue Protein Staining Solution (Thermo Fisher) and shake for 1 h.
  9. Wash 3 x 10 min in MQ water.
  10. The gel is now ready for imaging.

Author: Nyytti Koskinen

This protocol is based on:

This protocol has been modified mostly to be a larger scale attempt at protocol v.2.



Day 1: preculture

Start this protocol earliest at 15.45.

Note:Work in a laminar flow hood.

  1. Set up the laminar flow hood with inoculation loops and plates.
  2. Go get glycerol preps for strains you want to use for protein expression from -80 °C one at a time. Do not let the glycerol prep thaw. Keep glycerol prep on ice and take it back to the freezer as soon as possible. Remember to get the control strain (does not have expression plasmid inside) glycerol prep as well.
  3. Scrape of some frozen bacteria from the glycerol prep with a sterile inoculation loop in a laminar flow hood.
  4. Streak bacteria onto the LA plate with relevant antibiotics. The control strain may be plated to a LA plate without any antibiotics. Remember to take a new sterile inoculation loop for each streak. Make two plates per prep. The partner will use this time to take the prep back to the freezer and get the next one. Make one control plate by streaking with a sterile inoculation loop without adding any bacteria from glycerol preps.
  5. Incubate overnight at 37 °C. Take the plates to the incubator earliest at 16.


Day 2: Overnight liquid culture

Note:Work in a laminar flow hood.

  1. At 9: get the plates from the incubator. Put successful plates (single colonies) to the fridge and take unsuccessful ones to the GMO plate trash. The control plate is successful if nothing grows on it.
  2. Earliest at 21: Set the incubator at 37 °C. Take the plates from the fridge and take them to the laminar room.
  3. Pipette 4 mL of sterilized LB medium with 0.5 % D-glucose into 14 mL snap cap tubes. Add relevant antibiotics to media. Remember to mix the antibiotic before pipetting. Do not add antibiotics for the control strain tube! Make 2 precultures per strain.
  4. Add colony in a sterile manner using an inoculation loop to the tube.
  5. Grow cells for 18 hours at 37 °C 250 rpm. Remember to leave the tube tilted and the cap ajar. Put the plates in the fridge. Move IPTG from the freezer to the fridge for thawing.


Day 3: Expression

Start the protocol after 18 hours have passed since putting the overnight culture to the incubator so at about 16.

  1. Write the name of the protein on the 500 mL flasks. Add relevant antibiotics to the flasks. Remember to mix the antibiotic before pipetting. Do not add antibiotics for the control strain! You should have 4 x 500 mL flasks per strain.
  2. Blank the spectrophotometer with 1 mL of media.
  3. Add 1 mL of preculture to expression cultures. You have 2 x each protein preculture so add preculture from each to two expression medias. Put the rest of the preculture to the liquid GMO waste.
  4. Grow expression cultures at 37 °C with 250 rpm shaking until OD600=0.5-0.6. Measure OD600 in a sterile manner. Each measurement requires 1 mL of medium. You can reuse the cuvette, if you wash it with MQ water between measurements. Pour the media after measuring to liquid GMO waste. Remember that E. coli doubling time is about 20 minutes.
  5. Induce the expression by adding IPTG to a final concentration of 0.2 mM. For 100 mL of culture you need 0.2 mL of IPTG. While inducing, the partner will set the incubator at 26 °C. After inducing, take the IPTG back to the freezer.
  6. Grow the cultures at 26 °C with 250 rpm for 12 hours.


Day 4: periplasmic extraction

From this point, we are no longer working in a sterile manner.

We are doing periplasmic extraction to every culture, but purification only to 2 of the cultures for each protein.

  • Partner should use the time that goes into periplasmic extraction to recharge HisPur cobalt resin if necessary. Resin recharging is done according to this protocol.
  1. Get the TSE buffer (200 mM Tris-HCl pH 7.5, 500 mM sucrose, 1 mM EDTA) from the fridge to room temperature.
  2. Cool the centrifuge to 4 °C.
  3. Get the expression cultures from the shaker after 12 hours have passed so at about 8.
  4. Move the cultures to 50 mL tubes by pouring. Max 40 mL per tube, so three tubes are needed for 1 flask! Weight the tubes to find out the centrifuge pairs. The weights should not differ more than 0.1 g!
  5. Remember to add the centrifuge adapters! Spin the culture down at 8,000 x g, 4 °C for 20 min.
  6. Discard the supernatant by pouring to liquid GMO waste and tap on paper to get rid of as much of the liquid as possible. The paper goes to solid GMO waste.
  7. Weight the cells approximately.
  8. Carefully resuspend the pellet in 3 mL TSE (200 mM Tris-HCl pH 7.5; 500 mM sucrose, 1 mM EDTA) per gram of cells.
  9. Incubate the suspension at room temperature for 10 min.
  10. Cold-shock the cells by adding 3 mL of ice-cold MQ water per gram of cells. Combine the same protein cultures.
  11. Incubate the suspension on ice for 10 minutes.
  12. Spin the cells at 8,000 x g, 4 °C for 20 minutes. Collect the supernatants from each 50 mL tube to two 15 mL tubes. Keep on ice.
  13. Do Bradford assay for the periplasmic extractions on a 96-well plate. Add the Bradford reagent in a fume hood.
  14. Measure total protein concentration with Tecan Infinite plate reader.
  15. For 1 of the 2 15 mL tubes per each protein, add the same volume of 50 % glycerol to the tube in a sterile manner. Put these tubes in the freezer.


Day 4: IMAC purification

Keep the buffers on ice.

Buffer A: 20 mM sodium phosphate pH 7.5, 100 mM NaCl + 5 mM imidazole + 10 % glycerol

Buffer B: 20 mM sodium phosphate pH 7.5, 100 mM NaCl + 250 mM imidazole+ 10 % glycerol

  1. Pretreatment of the co-resin.
    • Take the used slurries for the correct proteins from the fridge.
    • Move the slurries from the 15 mL tubes to 50 mL tubes. 5 mL MQ water is added and the suspension is centrifuged 5 min 700 x g.
    • Remove the storage buffer on top with a pipette.
    • The pellet is resuspended in 10 mL of Buffer A (20 mM sodium phosphate pH 7.5; 100 mM NaCl + 5 mM imidazole + 10 % glycerol) and the resin is pelleted again 5 min 700 x g.
    • Remove the excess Buffer A on top with a pipette.
    • The pellet is resuspended in 10 mL of Buffer A (5 mM imidazole) again and the resin is pelleted again 5 min 700 x g.
    • Remove the excess Buffer A on top with a pipette. Keep pelleted resin on ice.
  2. Binding of the His-tagged protein.
    • Add 15 mL of buffer A to periplasmic extractions.
    • Add protein extraction (the ones without added glycerol) to the reaction tube.
    • The His-tagged protein is allowed to bind to the resin for 30-45 min with gentle mixing at 4 °C degrees.
    • Centrifuge for 15 min at 700 x g 4 °C degrees.
    • Remove the excess protein extraction with a pipette. Take a 1 mL sample from every flow-through in a 2 mL centrifuge tube. Do Bradford assay. After that, add 1 mL of 50 % glycerol in a sterile manner. Put them in -20 °C.
    • A second centrifugation of the supernatant may be necessary for effective pelleting of the resin.
  3. Washing out unbound protein.
    • The resin pellet is resuspended in a total volume about 10 mL of cold Buffer A and incubated with gentle shaking for about 10 min before centrifugation.
    • Centrifuge 5 min 700 x g 4 °C degrees.
    • Remove the buffer on top by pouring and tapping gently.
    • Repeat the wash process three times total.
  4. Elution.
    • Add 1 mL of Buffer B (20 mM sodium phosphate pH 7.5; 100 mM NaCl + 250 mM imidazole + 10 % glycerol).
    • Incubate 10 min 4 °C degrees with shaking.
    • Centrifuge 5 min at 700 x g 4 °C degrees.
    • Move the supernatant on top with a pipette to an 1,5 mL centrifuge tube. This supernatant contains the protein!
    • Repeat three times total.
    • Keep fractions that contain protein (based on Bradford assay) and add the same volume of 50 % glycerol in a sterile manner. Put it in -20 °C.
    • Analysis of fractions. Do Bradford assay for the fractions and on a 96-well plate. Add the Bradford in a fume hood.
    • Measure concentrations with plate reader.

At the end of the day, Bradford assay should have been performed on all fractions, flow-through and extractions.



Day 5: SDS-PAGE

Refer to the SDS-PAGE protocol for more details.

  1. Remove the comb and tape from the gels and assemble the electrophoresis cell.
  2. Fill the inner and outer buffer chambers with running buffer.
  3. Prepare the samples in the 2 mL centrifuge tube by adding 15 µL protein sample and 5 µL 4 x SLB.
  4. Heat the samples at 90-100 °C for 5 min in the heat block with water in the wells. Load 10 µL of the sample on the gel. Remember to load 2 ladders (3 µL each) as well.
  5. Connect the electrophoresis cell to the power supply and perform electrophoresis. Run conditions: 200 V
  6. After the run is complete, turn the power supply off and disconnect the electrical leads. Pop open the gel cassettes and remove the gel by floating it off the plate into water.
  7. Wash the gel in MQ for 10 minutes three times.
  8. Add PageBlue Protein Staining Solution (Thermo Fisher) and shake for 1 h.
  9. Wash 3 x 10 min in MQ water.
  10. The gel is now ready for imaging.

Author: Pekka Hyppölä

This protocol is based on:



  1. Strip the resin of cobalt ions by adding 5 mL of 0.2 M EDTA pH 7.0, incubate for 5 min in a shaker at 4 °C. Centrifuge for 2 minutes in 4 °C 700 x g. Remove the supernatant carefully with a pipette.
  2. Wash excess EDTA from the resin with 5 mL of MQ water. Centrifuge for 2 minutes in 4 °C 700 x g. Remove the supernatant carefully with a pipette.
  3. Charge the resin with 5 mL 50 mM CoCl2 solution, incubate for 5 min in a shaker at 4 °C. Centrifuge for 2 minutes in 4 °C 700 x g. Remove the supernatant carefully with a pipette.
  4. Wash resin with 5 mL of MQ water. Centrifuge for 2 minutes in 4 °C 700 x g. Remove the supernatant carefully with a pipette.
  5. Wash resin with 5 mL of 300 mM NaCl. Centrifuge for 2 minutes in 4 °C 700 x g. Remove the supernatant carefully with a pipette.
  6. Wash resin with 5 mL of MQ water. Centrifuge for 2 minutes in 4 °C 700 x g. Remove the supernatant carefully with a pipette.
  7. The resin is now ready to be used.


Culture-related protocols

Author: Nyytti Koskinen

This protocol is based on:



1. Measure 25 g of premixed LB formula per 1 L.

2. Add about 950 mL of MQ water and mix until powder is dissolved.

3. Adjust the volume to 1 L.

4. Autoclave using liquid cycle as soon as possible. If autoclave is not readily available, store at 4 °C.

Author: Nyytti Koskinen

This protocol is based on:



Note: Work in a laminar flow hood.

  1. Pipette in a sterile manner 800 µL of overnight culture to a sterile 1.5 mL centrifuge tube. Pipette 200 µL of 50 % sterile glycerol to the tube.
  2. Shake vigorously to mix. Label the tubes and store in the freezer (-80 °C).

Author: Nyytti Koskinen

This protocol is based on:



500 mL of LA per one sleeve of plates. Adjust the volumes according to the wanted amount of plates.

  1. Measure 25 g of premixed LB formula.
  2. Add about 950 mL of MQ water and mix.
  3. Adjust the volume to 1 L.
  4. Add 15 g of agar per 1 L.
  5. Autoclave. Store at 4°C if autoclave is not readily available.
  6. Allow to cool before adding antibiotics. LA should feel hot, but not burning hot. Suitable concentrations depend on the antibiotic, 50 µg/mL for kanamycin and 100 µg/mL for ampicillin for example.
  7. Pour plates into sterile Petri dishes in a laminar flow hood. Allow to dry for at least 15 minutes before closing the lid.
  8. Store in the fridge and use within two months.


Competent cells and transformation

Author: Nyytti Koskinen

This protocol is based on:



Note: Work in a laminar flow hood.

Preculture

  1. Pipette 4 mL of sterilized LB medium into a 14 mL snap cap tube. Add E. coli BL21(DE3)pLysS colony in a sterile manner using an inoculation loop to the tube.
  2. Grow cells for 16 hours at 37 °C 250 rpm. Remember to leave the tube tilted and the cap ajar.


Competent cells

  1. Prepare the broth and buffers.
    • Psi broth:
      • per liter: 5 g Bacto yeast extract, 20 g Bacto Tryptone, 5 g magnesium sulfate 7 H2O, pH 7.6 with potassium hydroxide
      • can be autoclaved
    • TfbI buffer:
      • Per 200 mL: 30 mM potassium acetate, 100 mM rubidium chloride, 10 mM calcium chloride 2 H2O, 50 mM manganese chloride, 15 % v/v glycerol, pH 5.8 with 10 % acetic acid
      • Has to be sterile filtered
    • TfbII buffer:
      • Per 100 mL: 10 mM MOPS, 75 mM calcium chloride, 10 mM rubidium chloride, 15 % v/v glycerol, pH 6.5 with diluted NaOH
      • Has to be sterile filtered
  2. Cool the centrifuge to 4 °C. This should be done at least an hour before step 6.
  3. Pipette 1 mL of overnight culture into 100 mL sterile Psi broth in 500 mL sterile flask.
  4. Incubate at 37 °C 200 rpm.
  5. Use the spectrophotometer in the laminar room. Measure OD550 regularly. Reset spectrophotometer with Psi broth at 550 nm. Take the first sample after 45 min. To take a sample pipette 1 mL of culture into a cuvette under sterile conditions. You can use the same cuvette for all measurements, just remember to wash it with MQ water. Let culture grow until 0.48. Remember that E. coli doubling time is 20 minutes!

    6. From this point - work on ice! Everything used must be on ice - even buffers and empty centrifuge tubes!

  6. Keep culture in ice for exactly 15 minutes. Bury it in ice.
  7. Use sterile 50 mL tubes. Centrifuge for 5 minutes 3,000 x g 4 °C.
  8. Discard the supernatant and add 0.4 of the original culture volume of sterile TfbI (here 100*0.4=40 mL, so 20 mL per tube!!!). Resuspend very gently using a 10 mL pipette. Incubate in ice for 15 minutes.
  9. Centrifuge for 5 minutes 3,000 x g 4 °C.
  10. Discard the supernatant and resuspend very carefully in 0.04 of the original volume of sterile TfbII (here 4 mL, so 2 mL per falcon!). Incubate for 15 minutes in ice.
  11. Aliquot into 50 µL aliquots in sterile 1.5 mL centrifuge tubes. One tube of competent cells is required for one transformation. Either use immediately or quick freeze in liquid nitrogen and store at -80 °C. Thaw on ice just before using in a transformation experiment. You cannot re-freeze the cells!!


Testing competence

  1. Transform cells with 0.01, 0.1 and 1 ng of plasmid DNA that is typical regarding insert size.
  2. Incubate plates in 37 °C for 16 hours exactly.
  3. Count the colonies. You can calculate the competence level with this calculator: “Bacteria Transformation Efficiency Calculator,” www.sciencegateway.org. https://www.sciencegateway.org/tools/transform.htm

Author: Nyytti Koskinen

This protocol is based on:



Materials:

  • 1.5 mL centrifuge tubes of competent E. Coli BL21(DE3)pLysS cells, made according to this protocol
  • Plasmid solutions
  • LB media, 950 µL per transformation
  • LA plates with relevant antibiotics, 2 per transformation + control


Remember to label tubes appropriately!

  1. Start warming up the shaker to 37 °C and heat block to 42 °C.
  2. Add competent BL21(DE3)pLysS cell tubes directly in ice!
  3. Thaw a tube of BL21(DE3)pLysS competent E. coli cells on ice for 10 minutes.
  4. Add 1–5 µL of plasmid solution to the cell mixture depending on the cell competence level. We used about 70 ng to 400 ng of plasmid DNA as our cell competence level was quite low. Keep cells on ice. Carefully flick the tube 4–5 times to mix cells and DNA. Do not keep off ice for more than 2-3 seconds. Do not vortex. Repeat for all tubes. For the control, skip this step.
  5. Incubate the mixture on ice for 10 minutes. Do not mix.
  6. Heat shock mixture on heat block with water added to the wells at exactly 42 °C for exactly 45 seconds. Do not mix.
  7. Place on ice for 2 minutes. Do not mix.
  8. Pipette 950 µL of room temperature LB into the mixture.
  9. Place at 37 °C 250 rpm for 30-60 minutes.
  10. Plate on LA plates with relevant antibiotics. For every transformation do 2 plates. On the first plate add 150 µL of the transformation mixture. After that, spin the rest of the mixture (850 µL), discard the supernatant, resuspend the pellet in as little liquid as possible and then plate that to the other plate.
  11. Incubate overnight at 37 °C.


As long as you got at least a single colony, the transformation was successful. If you didn’t get any colonies, you can redo the protocol with higher DNA amount or with better competent cells. It’s also worth it to check if the selection plate antibiotic and the antibiotic resistance gene in the plasmid match. If the plate ends up confluent, you can redo the transformation protocol by plating a lesser volume to the plates. If the control plate has any colonies, it usually means the antibiotic is not functional. This could be because the plate is too old and the antibiotic has degraded or the antibiotic was added while LA was still too hot.



Aging bloodstains

Author: Ari Heino

This protocol is based on:



The drying portion of this protocol didn’t work as planned. Our samples were hypothesized to have dried in about 4 hours but the drying ended up taking about 18 hours, after we turned the cotton swabs the soft end pointing up. We don’t know how long it might’ve taken for the samples to dry if they had been left in their tubes with the soft end pointing down. The Tris-HCl used in this protocol is too weak (10 mM) to effectively buffer against pH changes in blood. Please refer to the final version of the protocol.

The work must be done abiding to the rules explained here. Dedicated lab coats should be worn, buttoned up. All work should be performed in a fume cupboard dedicated solely for handling biological samples. Protective eyewear and gloves should be worn at all times. Gloves and used pipette tips should be disposed into a dedicated container. These same rules apply whenever handling blood samples and samples derived from blood.



Taking a sample from the blood in-case of exposure to the blood occurs

Before beginning the work with blood, take a 1 mL sample of the blood in case anyone is exposed to the blood and it’s necessary to measure what was in the blood the person was exposed to. Store the sample in -20 °C immediately. Dispose of this sample only after all the other blood samples have been disposed of.



Making the fresh blood samples

These samples will be used for comparative 0-day tests to measure how dried blood differs from blood that has never been dried.

  1. Move 3 x 120 µL of fresh blood to 2 mL tubes containing 310 µL of ice-cold MQ water.
  2. Allow to incubate on ice for 2 hours.
  3. Turn the tubes upside down 6 times and allow them to incubate another hour.
  4. Add 50 µL of pH 7.1 100 mM Tris-HCl with 1540 mM NaCl. Turn upside down 12 times. Store in the fridge until further processing.


Making the fresh plasma samples

  1. Add 200 µL of fresh blood to two 2 mL tubes.
  2. Add 300 µL of cold 3.9 M ammonium sulphate pH 7.1 to each tube.
  3. Nitrogenate the tubes according to the nitrogenation protocol and close the cap.
  4. Mix by gently turning the tubes upside down 12 times.
  5. Incubate at 5 °C for 1 hour on a tube shaker with a medium to low rpm.
  6. Centrifuge at 10,000 x g at 5 °C for 15 min.
  7. Move 250 µL of the fresh plasma sample to an empty 2 mL tube labeled “iGEM fresh plasma”. Store it in ice.
  8. Move 3 x 200 µL of fresh plasma samples to empty 2 mL tubes labeled "iGEM Preserved fresh plasma”.
  9. Nitrogenate the 3 tubes containing the preserved fresh plasma samples.
  10. Incubate the 3 tubes on ice for 2 hours, and move them to -20 °C afterwards.
  11. Process these samples according to the preserved fresh plasma HSA purification protocol.


Making the dried bloodstain samples

  1. Cut Q-tips so that they fit perfectly upright in 2 mL tubes.
  2. Move 120 µL blood sample to an empty 2 mL tube.
  3. Place a shortened Q-tip soft end pointing down on the blood sample.
  4. Turn the Q-tips upside down.
  5. Let the bloodstain dry for at least 4 hours or until fully dried.
  6. Age the blood stains in a designated drawer caps open to allow bloodstains to get exposed to air. Only open the drawer when taking out aged bloodstains.
  7. When the bloodstains have aged, nitrogenate and store the samples according to this protocol.

Author: Ari Heino

The work must be done abiding to the rules explained here. Dedicated lab coats should be worn, buttoned up. All work should be performed in a fume cupboard dedicated solely for handling biological samples. Protective eyewear and gloves should be worn at all times. Gloves and used pipette tips should be disposed into a dedicated container. These same rules apply whenever handling blood samples and samples derived from blood.



  1. Open the 2 mL tube containing the aged bloodstain and leave it slightly ajar.
  2. Blow nitrogen gas into the 2 mL sample tube from the mouth of the tube for 6 seconds.
  3. Close the caps while still blowing nitrogen gas on the samples.
  4. Store the samples in -21 °C.


nitrogen gas bottle

Nitrogen bottle during nitrogenation protocol.



Sped-up video depicting the protocol:

Author: Ari Heino

This protocol describes safe generation and storage of bloodstain samples for non-clinical testing. It streamlines our previous workflow. The only notable changes to stain generation were made to expedite drying. The bloodstain generation is adapted from Fresh blood handling protocol which also described long-term storage of frozen plasma; that content is omitted here.

Follow all local biosafety rules for handling potentially infectious biological materials.



Making the dried bloodstains

  1. Cut the cotton swab sticks just short enough to fit fully inside a 2 mL tube with the lid closed. Aim for a uniform length so the stick tip just touches the roof of the closed lid. Prefer wood or plastic sticks; cellulose may absorb water and alter the stain volume. From this point on, handle swabs with tweezers, not by hand.
  2. Pipette 120 µL of blood into each tube.
  3. Place a shortened cotton swab with the soft end pointing down into each tube.
  4. Allow the cotton swab to absorb the blood from the tube bottom for 30 min.
  5. Invert each swab so the soft end points up out of the tube.
  6. Dry for ~5 h under generous airflow. Confirm drying before storage by dissecting one stain; it should be dry at the core. After initial drying, age the samples in a controlled environment or store them as below.


Nitrogenation of aged bloodstains for storage

  1. Invert each swab so the soft end points down into its tube.
  2. Open the nitrogen line and set a modest flow.
  3. Crack the tube lid only enough to insert the nitrogen gas hose’s nozzle tip. Do not open so wide that gas flow could propel the swab out. Displace air in the tube by placing the nozzle tip slightly inside and flushing for 6 s.
  4. While gas is flowing, close the lid.


Hemoglobin-HSA separation

Author: Ari Heino

This protocol is based on:



Disclaimer: According to further research, we deemed the pH adjustment of the buffer to the pI of hemoglobin and HSA unnecessary. The buffering was also too weak. Most other HSA precipitation protocols using ammonium sulphate rely purely on changing the ammonium sulphate concentrations while keeping the pH stable. The ammonium sulphate concentrations used in this protocol are too low. See revised protocol here.



The work must be done abiding to the rules explained here. Dedicated lab coats should be worn, buttoned up. All work should be performed in a fume cupboard dedicated solely for handling biological samples. Protective eyewear and gloves should be worn at all times. Gloves and used pipette tips should be disposed into a dedicated container. These same rules apply whenever handling blood samples and samples derived from blood. The manipulation of the sample should be done in the dedicated fume hood. Incubations and centrifugations should be done in the cold room.



This is a method for purifying HSA by ammonium sulphate precipitation. This method is designed for samples that have been extracted according to bloodstain extraction for downstream purification protocol. The pH of the buffer used in the extraction method is already set at the pI of hemoglobin. In the first part, hemoglobin will be precipitated by bringing the ammonium sulphate concentration to about 50 %. Then the hemoglobin - along with any other undissolved debris - will be pelletted in by centrifugation. In the second part, pH will be lowered to the pI of HSA, and ammonium sulphate concentration will be increased further, to about 65 %. HSA will be pelleted and the supernatant will be discarded. After this, the HSA should be ready for further downstream processes.



Always remember to load the centrifuge symmetrically! Keep the caps of the tubes close unless you are adding things to it or taking things out.



Hemoglobin precipitation

  1. Add 480 𝛍L of cold 3.9M ammonium sulphate pH 7.1.
  2. Mix by turning the tube upside down 12 times. Allow the bubble to travel all the way up before flipping again.
  3. Incubate on a shaker, at a modest rpm for 15 min at 5 °C.
  4. Centrifuge 10,000 x g for 15 min at 5 °C.
  5. Collect the supernatant carefully to a separate 2 mL centrifuge tube with the pipette.


HSA precipitation

  1. Add 40 𝛍L of 150 mM citric acid cold pH 4.7.
  2. Add 500 𝛍L of cold 3.9 M ammonium sulphate pH 4.7 to the sample.
  3. Mix by turning the tube upside down 12 times. Allow the bubble to travel all the way up before flipping again.
  4. Incubate on a shaker, at a modest rpm for 15 min at 5 °C.
  5. Centrifuge 12,000 x g for 15 min at 5 °C.
  6. Collect the supernatant into a separate 2 mL tube. Remember to save the pellet as well.


The sample is now ready for downstream processing. Solvent should be selected according to the process. If the protocol doesn’t require specific solvent, 240 𝛍L of pH 7.1 1 x Tris-HCl should be used. Ions can be further scavenged with cold 80 % isopropanol before solvation, if required. For Bradford assay, use an equal amount of MQ water.

Author: Pekka Hyppölä

This protocol is based on:

  • V. A. Memoli and G. J. Doellgast, “Hemoglobin & serum albumin: Salt-mediated hydrophobic chromatography,” Biochemical and Biophysical Research Communications, vol. 66, no. 3, pp. 1011–1016, Oct. 1975, doi: https://doi.org/10.1016/0006-291x(75)90740-8
  • ‌ V. B. Jovanović, A. Z. Penezić-Romanjuk, I. D. Pavićević, J. M. Aćimović, and L. M. Mandić, “Improving the reliability of human serum albumin-thiol group determination,” Analytical Biochemistry, vol. 439, no. 1, pp. 17–22, Apr. 2013, doi: https://doi.org/10.1016/j.ab.2013.03.033
  • “Ammonium Sulfate Calculator from EnCor Biotechnology Inc.,” Encorbio.com, 2025. https://files.encorbio.com/protocols/AM-SO4.htm
  • ‌ W. I. Wood, “Tables for the preparation of ammonium sulfate solutions,” Analytical Biochemistry, vol. 73, no. 1, pp. 250–257, May 1976, doi: https://doi.org/10.1016/0003-2697(76)90165-2.


  1. Prepare ammonium sulphate stock (for 50 mL: 1.667 mL 1.5 M Tris-HCl pH 7.1, 36.5 g (NH4)2SO4 , 48,333 mL MQ water).
  2. Label 2 mL centrifuge tubes as shown below. AS stands for ammonium sulphate, the number stands for the ammonium sulphate concentration and S stands for supernatant.

    AS1-45

    AS1-54

    AS1-70

    AS1-S



    AS2-54

    AS2-60

    AS2-70

    AS2-S



    AS3-54

    AS3-70

    AS3-75

    AS3-S



    AS4-54

    AS4-70

    AS4-S

  3. Add 240 µL of the extracted 0dF samples into the lowest concentration tubes, so tubes AS1-45, AS2-54, AS3-54, AS4-54. You can find the extraction protocol here.


AS1-45 tube

  1. Into the AS1-45 tube, add 196 µL of cold ammonium sulphate stock.
  2. Mix by turning the tube upside down 12 times. Allow the bubble to travel all the way up before flipping again.
  3. Incubate on a shaker, at a modest rpm for 15 min at 5 °C.
  4. Centrifuge at 5,000 x g for 10 min at 5 °C.
  5. Collect the supernatant carefully to the tube AS1-54 with a pipette.


AS2-54, AS3-54, and AS4-54 tubes

  1. Into AS2-54, AS3-54, and AS4-54 tubes, add 282 µL of cold ammonium sulphate stock.
  2. Mix by turning the tube upside down 12 times. Allow the bubble to travel all the way up before flipping again.
  3. Incubate on a shaker, at a modest rpm for 15 min at 5 °C.
  4. Centrifuge at 5,000 x g for 10 min at 5 °C.
  5. Collect the supernatant carefully to the following tubes with a pipette:
    • AS2-54 → AS2-60
    • AS3-54 → AS3-70
    • AS4-54 → AS4-70


AS1-54 tube

  1. Into the AS1-54 tube, add 85 µL of cold ammonium sulphate stock.
  2. Mix by turning the tube upside down 12 times. Allow the bubble to travel all the way up before flipping again.
  3. Incubate on a shaker, at a modest rpm for 15 min at 5 °C.
  4. Centrifuge at 5,000 x g for 10 min at 5 °C.
  5. Collect the supernatant carefully to tube AS1-70 with a pipette.


AS2-60 tube

  1. Into the AS2-60 tube, add 78 µL of cold ammonium sulphate stock.
  2. Mix by turning the tube upside down 12 times. Allow the bubble to travel all the way up before flipping again.
  3. Incubate on a shaker, at a modest rpm for 15 min at 5 °C.
  4. Centrifuge at 5,000 x g for 10 min at 5 °C.
  5. Collect the supernatant carefully to tube AS2-70 with a pipette.


AS1-70, AS3-70 and AS4-70 tubes

  1. Into AS1-70, AS3-70, and AS4-70 tubes, add 278 µL of cold ammonium sulphate stock.
  2. Mix by turning the tube upside down 12 times. Allow the bubble to travel all the way up before flipping again.
  3. Incubate on a shaker, at a modest rpm for 15 min at 5 °C.
  4. Centrifuge at 8,000 x g for 10 min at 5 °C.
  5. Collect the supernatant carefully to the following tubes with a pipette:
    • AS1-70 → AS1-S
    • AS3-70 → AS3-75
    • AS4-70 → AS4-S


AS2-70 tube

  1. Into AS2-70, add 200 µL of cold ammonium sulphate stock.
  2. Mix by turning the tube upside down 12 times. Allow the bubble to travel all the way up before flipping again.
  3. Incubate on a shaker, at a modest rpm for 15 min at 5 °C.
  4. Centrifuge at 8,000 x g for 10 min at 5 °C.
  5. Collect the supernatant carefully to tube AS2-S with a pipette.


AS3-75 tube

  1. Into the AS3-75 tube, add 160 µL of cold ammonium sulphate stock.
  2. Mix by turning the tube upside down 12 times. Allow the bubble to travel all the way up before flipping again.
  3. Incubate on a shaker, at a modest rpm for 15 min at 5 °C.
  4. Centrifuge at 8,000 x g for 10 min at 5 °C.
  5. Collect the supernatant carefully to tube AS3-S with a pipette.


Resuspend the pellets with 240 µL of MQ water using a pipette.

Author: Ari Heino

This protocol is based on:

  • R. F. Rieder, “Hemoglobin stability: observations on the denaturation of normal and abnormal hemoglobins by oxidant dyes, heat, and alkali,” Journal of Clinical Investigation, vol. 49, no. 12, pp. 2369–2376, Dec. 1970, doi: https://doi.org/10.1172/jci106456


  1. Extract 3 x 1dF bloodstain samples according to this protocol.
  2. Set the heat block to 60°C. Add 200 μL of MQ water to wells.
  3. Prepare 12 x 2 mL tubes and label them according to the table below.
    Sample name Time on the heat block
    0dFE - H1 5 sec
    0dFE - H2 10 sec
    0dFE - H3 20 sec
    0dFE - H4 30 sec
    0dFE - H5 1 min
    0dFE - H6 2 min
    0dFE - H7 3 min
    0dFE - H8 5 min
    0dFE - H9 10 min
    0dFE - H10 20 min
    0dFE - H11 30 min
    0dFE - H12 60 min
  4. Add 365 μL of pH 7.1 10 mM Tris-HCl with 154 mM NaCl to each sample tube.
  5. Add 15 μL of 1.5 M Tris-HCl pH 7.1 to each sample tube.
  6. Add 100 μL of saturated ammonium sulphate, pH adjusted to 7.1 with Tris-base, to each sample tube.
  7. Add 120 μL of 0dFE sample to each sample tube.
  8. Mix samples by turning them upside down 12 times.
  9. Place samples on the heat block. After they have been on the heat block for the correct amount of time (refer to the table above), move them to the refrigerator.
  10. After the samples have incubated in the fridge for at least 15 minutes, centrifuge 12,000 x g 15 min 5 °C.
  11. Measure hemoglobin peaks at 420 nm and 570 nm. Measure HSA concentration using bromocresol green. Measure total protein concentration using Bradford assay. You can find related protocols here.

Author: Ari Heino

This protocol is based on:



Disclaimer: This is one of our earlier HSA purification protocols. Buffering in this protocol is too weak, and the ammonium sulphate concentrations added are too low. It is unlikely that this protocol could be used to successfully salt out HSA as-is. To precipitate HSA, ammonium sulphate concentration should be above 70% saturation (see referenced article).



The work must be done abiding to the rules explained here. Dedicated lab coats should be worn, buttoned up. All work should be performed in a fume cupboard dedicated solely for handling biological samples. Protective eyewear and gloves should be worn at all times. Gloves and used pipette tips should be disposed into a dedicated container. These same rules apply whenever handling blood samples and samples derived from blood. The manipulation of the sample should be done in the dedicated fume hood. Incubations and centrifugations should be done in the cold room.



While with the dried blood samples and with the lysed fresh blood samples, the hemoglobin was removed after hemolysis, the goal of the preserved fresh plasma samples is to try to remove whole erythrocytes before hemolysis can occur.



  1. Thaw the frozen preserved fresh plasma sample overnight in the refrigerator.
  2. Add 1/4 of the sample volume of pH 4.7 0.5 M citrate buffer to the sample.
  3. Add twice the amount of the added citrate buffer of ammonium sulphate 3.9 M pH 4.7.
  4. Mix by turning the tube upside down 12 times. Allow the bubble to travel all the way up before flipping again.
  5. Incubate on a shaker, at a medium to high rpm for 15 min at 5 °C.
  6. Centrifuge 12,000 x g for 15 min at 5 °C.
  7. Move the supernatant to a separate 2 mL tube. Take it to the fridge.
  8. The pellet is now ready for downstream processing. Solvent should be selected according to the process. If a protocol doesn’t require a specific solvent, 200 µL of pH 7.1 10 mM Tris-HCl with 154 mM NaCl should be used. Ions can be further scavenged with cold 80% isopropanol before solvation, if required. For Bradford, use an equal amount of MQ water.

Author: Ari Heino

This protocol is based on:

  • R. K. Mitra, S. S. Sinha, and S. K. Pal, “Hydration in Protein Folding: Thermal Unfolding/Refolding of Human Serum Albumin,” Langmuir, vol. 23, no. 20, pp. 10224–10229, Sep. 2007, doi: doi: https://doi.org/10.1021/la7014447.
  • ‌ K. Yamasaki and Makoto Anraku, “Stability of Albumin and Stabilization of Albumin Preparations,” Springer eBooks, pp. 25–49, Jan. 2016, doi: https://doi.org/10.1007/978-981-10-2116-9_2.
  • ‌R. K. Scopes, Protein Purification. Springer Advanced Texts in Chemistry, 1994.


This protocol is the final version of our hemoglobin-HSA separation method. The modifications made are based on experimentation by the ABOA 2025 team. The experimentation has been done utilizing the engineering cycle. Earlier versions for hemoglobin-HSA separation contained redundant steps. This protocol combines pH manipulation, ammonium sulphate precipitation, and selective heat treatment.



To increase the rate and likelihood of protein precipitation and aggregation, the pH is adjusted near the isoelectric point (pI) of the protein being removed. Ammonium sulphate causes “salting out” and enhances hydrophobic interactions between proteins, promoting aggregation. Heat treatment increases molecular motion and accelerates aggregation.



The protocol may be adapted to separate other proteins, but the pI, heat tolerance, and salt tolerance of the target protein must be considered. Ammonium sulphate precipitation is generally regarded as gentle and reversible. In the case of HSA, heating to 60 °C does not irreversibly denature it, as confirmed by literature and our nanoDSF experiments.



Important note: The pH should be 7.1 during heat treatment and lowered to 4.7 during HSA enrichment. During our experiments, only pH paper was available for use with the blood samples. Titration tests with a pH meter were performed without the blood matrix. It is worth confirming that the pH truly reaches 4.7 during HSA enrichment, as deviations could affect HSA solubility and yield.



Preparing saturated ammonium sulphate solutions

  1. Heat 50 mM pH 7.1 Tris-HCl and 200 mM pH 4.7 citrate buffer on a hotplate with vigorous (but not maximum) stirring. Do not boil.
  2. Add ammonium sulphate to ~5.5 M until it no longer dissolves. An increase in solution volume is expected.
  3. Remove from the hotplate and transfer to airtight containers.
  4. Cool at room temperature, then store at 4 °C overnight. Crystals should appear by the next day, confirming saturation.


Heat Treatment of Samples

The starting material is reconstituted bloodstain samples, prepared according to the final bloodstain generation and extraction protocols. These are initially diluted 1:4 compared to whole blood. Adding Tris/NaCl buffer and saturated ammonium sulphate solutions further dilutes the samples By the time the sample reaches the heating stage, the overall dilution is approximately 1:20 compared to whole blood. This dilution ratio is critical: if 1:4 reconstituted blood samples were heated directly, not only hemoglobin but also HSA and many other proteins would likely aggregate. Maintaining the lower protein concentration promotes selective aggregation of hemoglobin while keeping HSA soluble.



  1. Add 380 µL of 154 mM NaCl with 50 mM Tris-HCl (pH 7.1) to a 2 mL tube.
  2. Add 100 µL of previously prepared ammonium sulphate Tris pH 7.1.
  3. Add 120 µL of reconstituted blood sample.
  4. Mix by gently inverting the tube 12 times. Allow the bubble to travel the full length each time. Do not shake.
  5. Preheat the heating block to 60 °C. Add MQ water to wells for proper heat conduction.
  6. Place the tubes into the heat block wells and incubate for 115 min.
  7. Cool at room temperature for 15 min.
  8. Cool at 4 °C for 30 min.
  9. Centrifuge at 12,000 × g for 15 min at 4 °C.
  10. Carefully transfer the supernatant to a new 2 mL tube, avoiding disturbance of the pellet and aggregated protein plaques.


HSA Enrichment

  1. Add 10 µL of 800 mM citrate buffer (pH 3) to the 2 mL containing the supernatant. Rinse the pipette tip with the supernatant to ensure complete transfer.
  2. Add 1030 µL of previously prepared ammonium sulphate citrate solution pH 4.7.
  3. Mix by gently inverting 12 times.
  4. Incubate at 4 °C on a shaker at low rpm for 15 min.
  5. Centrifuge at 8,000 × g for 10 min at 4 °C.
  6. Discard the supernatant by pouring, then gently tapping against a paper towel.
  7. Dissolve the pellet in 120 µL of 50 mM Tris-HCl, pH 7.1.


Extracting a dried bloodstain

Author: Ari Heino



The work must be done abiding to the rules explained here. Dedicated lab coats should be worn, buttoned up. All work should be performed in a fume cupboard dedicated solely for handling biological samples. Protective eyewear and gloves should be worn at all times. Gloves and used pipette tips should be disposed into a dedicated container. These same rules apply whenever handling blood samples and samples derived from blood. The manipulation of the sample should be done in the dedicated fume hood. Incubations and centrifugations should be done in the cold room.



The caps should be kept closed unless you are adding things into the tubes or taking things out of them.



In this protocol, you have 3 x 2 mL centrifuge tubes per bloodstain. Tube 1 is the tube which contains the original bloodstain. The bloodstain extraction steps are done in tubes 1 and 2, and the extracted bloodstain is collected to tube 3.



Bloodstain extraction

  1. Rehydrate the bloodstain by adding 120 µL of 77 mM NaCl solution to tube 1. Incubate 10 min in room temperature.
  2. Prepare microcentrifuge tube 2 by adding 120 µL of 154 mM NaCl solution. Move the cotton swab with the rehydrated sample to tube 2. Incubate on a shaker with low rpm for 10 min.
  3. Add 120 µL of 154 mM NaCl solution to tube 1 and move the cotton swab from tube 2 to tube 1. Incubate on a shaker with low rpm for 10 min. Collect the fluid from tube 1 to tube 3.
  4. Add 120 µL of 154 mM NaCl solution to tube 2 and move the cotton swab from tube 1 to tube 2. Incubate on a shaker with low rpm for 10 min. Collect the fluid from tube 2 to tube 3.
  5. Move the cotton swab from tube 2 to tube 1, this time with the soft end of the cotton swab pointing up. Centrifuge at 500 x g for 1 min. Collect the fluid into tube 3.
  6. Discard the cotton swab and the tubes 2 and 1 into biological waste.

Author: Ari Heino



The work must be done abiding to the rules explained here. Dedicated lab coats should be worn, buttoned up. All work should be performed in a fume cupboard dedicated solely for handling biological samples. Protective eyewear and gloves should be worn at all times. Gloves and used pipette tips should be disposed into a dedicated container. These same rules apply whenever handling blood samples and samples derived from blood. The manipulation of the sample should be done in the dedicated fume hood. Incubations and centrifugations should be done in the cold room.



The caps should be kept closed unless you’re taking things out of the tubes or adding things into them.



This protocol discusses the use of “pH 7.1 Tris-HCl solutions. Our 10x pH 7.1 Tris-HCl-solution was 100 mM Tris-HCl + 1540 mM NaCl. 1x was 10 mM Tris-HCl + 154 mM NaCl, 0.5x was 5 mM Tris-HCl + 75.2 NaCl.



In this protocol, you have 3 x 2 mL centrifuge tubes per bloodstain. Tube 1 is the tube which contains the original bloodstain. The bloodstain extraction steps are done in tubes 1 and 2, and the extracted bloodstain is collected to tube 3.



Note: Keep the samples cold.

  1. Rehydrate the bloodstain by adding 120 µL of 0.5x pH 7.1 Tris-HCl solution to tube 1. Incubate 10 min in 4 °C.
  2. Prepare microcentrifuge tube 2 by adding 120 µL of 1x pH 7.1 Tris-HCl solution.
  3. Move the cotton swab with the rehydrated sample to tube 2. Incubate on a shaker with low rpm in 4°C for 10 min.
  4. Add 120 µL of 1x pH 7.1 Tris-HCl solution to tube 1.
  5. Move the cotton swab to tube 1. Incubate on a shaker with low rpm in 4 °C for 10 min.
  6. Collect the fluid from tube 2 to tube 3.
  7. Add 120 µL of 1x pH 7.1 Tris-HCl solution to tube 2.
  8. Move the cotton swab to tube 2. Incubate on a shaker with low rpm in 4 °C for 10 min.
  9. Collect the fluid from tube 1 to tube 3.
  10. Move the cotton swab from tube 2 to tube 1, this time with the soft end pointing up. Centrifuge at 500 x g for 1 min. Collect the separated fluid into tube 3.
  11. Store the sample in the refrigerator.
  12. Discard the cotton swab and the tubes 2 and 1 into biological waste.

Author: Ari Heino



The work must be done abiding to the rules explained here. Dedicated lab coats should be worn, buttoned up. All work should be performed in a fume cupboard dedicated solely for handling biological samples. Protective eyewear and gloves should be worn at all times. Gloves and used pipette tips should be disposed into a dedicated container. These same rules apply whenever handling blood samples and samples derived from blood. The manipulation of the sample should be done in the dedicated fume hood. Incubations and centrifugations should be done in the cold room.



The caps should be kept closed unless you’re taking things out of the tubes or adding things into them.



This protocol discusses the use of “pH 7.1 Tris-HCl solutions. Our 10x pH 7.1 Tris-HCl-solution was 100 mM Tris-HCl + 1540 mM NaCl. 1x was 10 mM Tris-HCl + 154 mM NaCl, 0.5x was 5 mM Tris-HCl + 75.2 NaCl.



In this protocol, you have 3 x 2 mL centrifuge tubes per bloodstain. Tube 1 is the tube which contains the original bloodstain. The bloodstain extraction steps are done in tubes 1 and 2, and the extracted bloodstain is collected to tube 3.



Note: Keep the samples cold.

  1. Label the tube 2 as x.2 (x is the name of the original sample, for example if the sample name is 0dF, name the tube 0dF.2). Label tube 3 as xE ( 0dF -> 0dFE). The original sample tube with the cotton swab is referred to as tube 1.
  2. Prepare microcentrifuge tube x.2 by adding 120 µL of 1x pH 7.1 Tris-HCl solution and 120 µL of 0.5x pH 7.1 Tris-HCl solution.
  3. Move the cotton swab to tube x.2. Incubate on a shaker with low rpm in 4 °C for 20 min.
  4. Add 240 µL of 1x pH 7.1 Tris-HCl solution to tube 1.
  5. Move the cotton swab from tube x.2 to tube 1. Incubate on a shaker with low rpm in 4 °C for 30 min.
  6. Collect the fluid from tube x.2 to tube xE.
  7. Move the cotton swab from tube 1 to tube x.2, this time with the soft end pointing up. Centrifuge at 500 x g for 1 min. Collect the fluid from tube x.2 and 1 to tube xE.
  8. Store the sample in tube xE in the refrigerator.
  9. Discard the cotton swab and the tubes x.2 and 1 into biological waste.

Author: Ari Heino



This protocol describes safe extraction of dried bloodstain samples for non-clinical testing. It streamlines our previous workflow. The extraction section is adapted from this protocol, with a different solvent to simplify downstream processing.



Follow all local biosafety rules for handling potentially infectious biological materials.



Extracting the bloodstains

For each bloodstain, label two 2mL tubes: tube 1 for extraction and tube 2 for pooled extract. Keep tubes closed unless you are handling the contents.

  1. To tube 1, add 240 µL of 154 mM NaCl, 50 mM Tris-HCl, pH 7.1.
  2. Remove the cotton swab from its storage tube and place it into tube 1 soft end down. Save the storage tube.
  3. Incubate 20 min at 4 °C on a shaker low rpm.
  4. Into the storage tube, add 240 µL of 154 mM NaCl, 50 mM Tris-HCl, pH 7.1.
  5. Move the cotton swab from tube 1 to the storage tube soft end down.
  6. Incubate 30 min at 4 °C low rpm.
  7. Transfer the extract from tube 1 to tube 2.
  8. Move the cotton swab from the storage tube to tube 1 soft end up.
  9. Centrifuge 1 min at 500 × g 4 °C.
  10. Discard the cotton swab appropriately.
  11. Transfer the liquids from tube 1 and the storage tube to tube 2.

At this point, all reconstituted 1:4 bloodstains should be pooled in tube 2.



Spectrophotometric measurements for blood samples

Author: Pekka Hyppölä

This protocol is based on:



Making bromocresol green reagent

  1. Mix 100 mL of 90 mM succinate buffer pH 4.2 with 0.01814826 g of bromocresol green.
  2. Adjust pH to 4.3.


Measurements with the spectrophotometer

  1. In a cuvette, mix 1.5 mL of reagent and 7.5 µL of sample or HSA standard.
  2. Incubate for 10 min at room temperature.
  3. Measure with a spectrophotometer at 630 nm. The sample remains stable for 60 minutes after mixing.
  4. If the sample is not within linear range, it can be diluted with 9 g/L NaCl.

Author: Ari Heino

This protocol is based on:



Disclaimer: Our methods were adapted for using our own specific bromocresol green reagent which was made using bromocresol green that was about 100 years old. This reagent did not give a linear response as it should have and had a very high background absorbance. Try to adapt your own protocol from the cited commercial protocols before considering following this one. If anything can be learned from this protocol, it is that if your spectrophotometric background reading is so high that it desensitizes your detector, you can experiment on substituting some of your sample-reagent matrix with clear solvent such as 0.9% NaCl or MQ water.



  1. Take the reagents to room temperature 30 minutes before use.
  2. Pipette 750 µL of 155 mM NaCl into a 2 mL centrifuge tube.
  3. With the pipette tip touching the solution, add 20 µL of sample to the 2 mL tube. Keeping the tip in the solution, press the plunger fully down and release fully up twice to rinse and ensure complete transfer. Keep the plunger fully down as you withdraw the tip.
  4. Pipette 730 µL of 0.26 mM bromocresol green reagent pH 4.3 into the 2 mL tube.
  5. Vortex and incubate at 37 °C for at least 10 minutes.
  6. Prepare the blank sample by mixing 770 µL of 155 mM NaCl with 730 µL of x M bromocresol green reagent pH 4.3. Blank the spectrophotometer at 630 nm with the blank sample.
  7. Measure the samples at 630 nm. Remember to always change the cuvette between measurements.

Author: Ari Heino

This protocol is based on:



We ended up never using the Bradford-assay portion of this protocol and instead adapted a new protocol from Bio-Rad’s Bradford Protein Assay protocol (https://www.bio-rad.com/webroot/web/pdf/lsr/literature/LIT33.pdf), which uses a more concentrated version of Bradford reagent and is more suitable for more diluted samples. We did discover that hemoglobin absorbs light very strongly at 420 nm. You can find the revised version of this protocol here.



The work must be done abiding to the rules explained here. Dedicated lab coats should be worn, buttoned up. All work should be performed in a fume cupboard dedicated solely for handling biological samples. Protective eyewear and gloves should be worn at all times. Gloves and used pipette tips should be disposed into a dedicated container. These same rules apply whenever handling blood samples and samples derived from blood. The manipulation of the sample should be done in the dedicated fume hood. Incubations and centrifugations should be done in the cold room.



In this method two types of samples are described: purified and extracted. Purified samples are any samples in which HSA has been purified using ammonium sulphate, such as lysed, purified samples, dried purified samples or purified plasma. Extracted samples refer to samples in which hemoglobin is still present.



Bradford assay

  1. Prepare BSA solutions from 0.2 to 0.9 mg/mL.
  2. Dilute the extracted samples so that they are in the range of the BSA standard’s concentration.
  3. Add 20 µL of your sample to a 2 mL tube.
  4. Add 980 µL of MQ-water to the same tube.
  5. Mix the tubes by vortexing quickly (1 second max) on lowest rpm. Remember correct vortexing technique: hold the tube from the top!
  6. Turn the tubes upside down 6 times.
  7. Add 500 µL of Bradford reagent to each dilution. Mix by turning the tube upside down 12 times.
  8. Incubate for 5 min.
  9. Start by measuring the blank at 595 nm. After that, measure the BSA standards, and lastly, your samples.


Measuring hemoglobin absorbance peaks

Remember to store these samples on ice!

  1. Dilute the extracted samples by adding 10 µL or 5 µL of the sample to 990 µL MQ water in a 2 mL tube.
  2. Dilute the purified samples by adding 15 µL or 10 µL of sample to 985 µL of MQ water in a 2 mL tube.
  3. Measure the absorbance at 420 nm.
  4. Hemoglobin at peak value of 420 nm can be quantified by using Beer-Lambert Law and molar extinction coefficient of 407,560 M-1 cm-1.

Author: Ari Heino, Nea Åberg

This protocol is based on:



The work must be done abiding to the rules explained here. Dedicated lab coats should be worn, buttoned up. All work should be performed in a fume cupboard dedicated solely for handling biological samples. Protective eyewear and gloves should be worn at all times. Gloves and used pipette tips should be disposed into a dedicated container. These same rules apply whenever handling blood samples and samples derived from blood. The manipulation of the sample should be done in the dedicated fume hood. Incubations and centrifugations should be done in the cold room.



In this method, we’ll be handling 3 different types of samples of bloodstains extracted according to this protocol: the dissolved large pellets from the second purification step, the small dissolved pellets from the first purification and whole blood samples.



The spectrophotometric measurements will be performed with Lambda Bio 40 UV/Vis Spectrometer (PerkinElmer Instruments). Hemoglobin must be measured on 420 nm (primary absorbance peak), and also may optionally be measured on 570 nm (secondary peak). The primary peak is more sensitive while the secondary peak may provide a more accurate measurement of the total hemoglobin. Bradford assay measurements shall be performed at 595 nm. The FE samples should be diluted in 154 mM NaCl and samples for downstream purification should be diluted in pH 7.1 10 mM Tris-HCl + 154 mM NaCl to allow for accurate measurements.



Bradford assay

  1. Prepare BSA dilutions for the standard from 1.2 to 10 µg/mL.
  2. Prepare a blank sample from pH 7.1 10 mM Tris-HCl + 154 mM NaCl.
  3. Add 500 µL of MQ water to diluted biological samples.
  4. Add 800 µL of samples to 2 mL tubes.
  5. Add 200 µL of Bio-Rad Protein Assay Dye Reagent Concentrate.
  6. Vortex immediately and incubate for 5 min. Measure within 1 hour of adding the reagent.
  7. Blank the spectrophotometer with pH 7.1 10 mM Tris-HCl + 154 mM NaCl. Measure the samples at 595 nm.


Hemoglobin absorbance peak measurements

  1. Add 1400 µL of MQ water to samples.
  2. Prepare a blank sample from 100 µL 1 x pH 7.1 Tris-HCl.
  3. Add 100 𝛍L of the appropriate sample the corresponding tube.
  4. Vortex and measure the absorbance at 420 nm and 570 nm. Remember to blank the spectrophotometer with pH 7.1 10 mM Tris-HCl + 154 mM NaCl.
  5. Hemoglobin at peak value of 420 nm can be quantified by using Beer-Lambert Law and molar extinction coefficient of 407,560 M-1 cm-1.

Author: Pekka Hyppölä

This protocol is based on:



Making 50 mL of 1 mM Ellman’s Reagent pH 8.0 solution

  1. Dilute 19.817 mg DTNB in a flask with about 40 mL of 0.1 M phosphate buffer pH 8.0.
  2. Adjust pH to 8.0.
  3. Pour into a 50 mL volumetric flask. Add 0.1 M phosphate buffer pH 8.0 to the 50 mL line. Label as “1 mM Ellman’s Reagent in 0.1 M phosphate buffer pH 8.0”.


Measuring free sulfhydryl concentration with Ellman’s assay

  1. Add 1350 µL of 0.1 M phosphate buffer pH 8.0 into a 2 mL centrifuge tube.
  2. Add 75 µL of the sample.
  3. Add 75 µL of the 1 mM Ellman’s Reagent pH 8.0 solution.
  4. Mix well and incubate at room temperature for 15 minutes for most samples, 8-24 h for HSA.
  5. Blank the spectrophotometer with 0.1 M phosphate buffer pH 8.0 at 412 nm, and measure absorbances of the samples.


Note: High concentrations (>1 mM) of free sulfhydryl may decrease the accuracy of the measured sulfhydryl concentration due to its higher absorbance values.



SDS-PAGE

Author: Tytti Sandholm

This protocol is based on:



Remember to quantitate the sample and find out the sample concentration. For SDS-PAGE, dilute or concentrate the protein to >0.5 mg/mL, the concentration should not be lower than that.

  1. Prepare the buffers
    1. Running buffer:
      • 10x SDS-PAGE (1 L)
      • (250 mM Tris, 1.92 M glycine, 1% SDS, pH 8.3)
    2. 4 x Sample Loading Buffer (SLB):
      • (0.250 M pH 6.8 Tris-HCl, 8 % SDS, 40 % glycerol, 5.78 mM bromophenol blue, 20 % 2-mercapto-ethanol)
  2. Prepare Bio-Rad’s Mini-PROTEAN TGX Gels and assemble the electrophoresis cell:
    1. Remove the comb and tape from the gels and assemble the electrophoresis cell.
    2. Fill the inner and outer buffer chambers with running buffer. Fill the upper (inner) buffer of each core with about 200 mL of 1x running buffer. Fill the lower (outer) buffer chamber to the indicator mark for 2 gels (550 mL) or 4 gels (800 mL) with 1x of running buffer. At runs with >200 V, fill the outer buffer chambers to the 4-gel mark.
  3. Prepare samples as indicated in the table below. Note that sample protein concentration should be >0.5 mg/ml.
    4. Protein sample 15 µL
    4 x Sample Loading Buffer 5 µL
    Total volume 20 µL
    Volume loaded to gel 10 µL
  4. Heat samples at 90-100 ℃ for 5 min.
  5. Load 10 µL of the sample on the gel. And remember to load the ladder!
    • For the ladder: 3 µL for Precision Plus Protein Dual Color Standard (Bio-Rad) or Color Prestained Protein Standard (New England Biolabs).
  6. Connect the electrophoresis cell to the power supply and perform electrophoresis according to the following conditions:
    • Run conditions: 125-200 V depending on the amount of gels

    7. After electrophoresis is complete, turn the power supply off and disconnect the electrical leads. Pop open the gel cassettes and remove the gel by floating it off the plate into water.

  7. Stain and image the gel.
    1. Wash gels three times for 10 minutes in MQ water 50 rpm.
    2. Add PageBlue Protein Staining Solution (Thermo Fisher) and let the gel stain for 1 hour 50 rpm.
    3. Wash gels three times for 10 minutes in MQ water 50 rpm.
    4. Image the gel with Gel Doc EZ Imager (Bio-Rad) using the white tray.