DNA Constructs
Figure 1: Cutting of pAG304GAL-ccdB and pAG304GPD-ccdB
Figure 1. Agarose gel electrophoresis of restriction digestion products. (1 % agarose in 1× TAE buffer, stained with EtBr). Vectors pAG304GAL-ccdb and pAG304GPD-ccdB cut with different restriction enzymes. Gene Ruler 1kb Ladder is used as a size marker. Control is the uncut vector.
Figure 1 shows Agarose gel electrophoresis which the presence of the empty pAG-304GAL-ccdB and pAG304GPD-ccdB vector at the size of 6.7 kb and the successful cutting with the enzymes Bsu361 and Xho1. Cutting with BstX1 and Not1 show unwanted cutting patterns. 1kb Ladder is used as a size marker. Control is the uncut vector. The successful cutting with Bsu361 and Xho1 indicates that these enzymes can be used for further cloning steps, while the unwanted cutting patterns with BstX1 and Not1 suggest that these enzymes are not suitable for our purposes. By using the enzymes Bsu361 and Xho1 for further cloning steps, we can ensure that our constructs are assembled correctly and efficiently. The presence of the empty vector at the correct size also confirms that our plasmid preparation was successful and that we have a good starting point for cloning a conditional mCherry under a GAL-Promoter for our first RNA target. The pAG304 vector is a genomically integrating vector to ensure the yeast strain does not lose the plasmid.
Figure 2: Cutting of pAG304-mCherry
Figure 2. Agarose gel electrophoresis of restriction digestion products. (1 % agarose in 1× TAE buffer, stained with EtBr). Vector pAG304-mCherry cut with the restriction enzymes Mfe1 and Bsu361. 1kb Ladder is used as a size marker. Control is the uncut vector.
Figure 2 shows an agarose gel electrophoresis of plasmid DNA obtained from miniprep of successfully transformed E. coli colonies showing a band corresponding to the expected size of the construct pAG304-ymCherry (6,4kb). The ymCherry was previously successfully cloned into the vector. The plasmid carries the mCherry gene cloned under control of a conditional GAL promoter. Plasmids were subsequently sent for sequencing, which confirmed the correct sequence of the construct. The yeast strain containing this construct was used for further integrating our TRAPS system to target the mCherry RNA.
Figure 3: Cutting of pAG305
Figure 3. Agarose gel electrophoresis of isolated pAG305. (1 % agarose in 1× TAE buffer, stained with EtBr). Vector pAG305 after cutting with SacI and MluI. 1kb Ladder is used as a size marker.
Figure 3 confirm the successful isolation of the pAG305 vector without the ccdb cassette, which is essential for our cloning and transformation strategy. pAG305 is a genomically integrating vector that allows for stable integration of our genes of interest into the yeast genome. The presence of a clear band at the expected size of 5,5 kb indicates that the vector was correctly cut. Bands visible at ~1,6kb and ~1kb confirm the cut out fragment (combined size of ~2,6kb corresponding to the size of the cut out including the ccdb cassette).
Figure 4: Cutting of pAG416
Figure 4. Agarose gel electrophoresis of isolated pAG416. (1 % agarose in 1× TAE buffer, stained with EtBr). Vector pAG416 after being cut with SacI and MluI. 1kb Ladder is used as a size marker.
Figure 4 confirms the successful isolation of the pAG416 vector. The pAG416 vector is a high-copy plasmid that allows for strong expression of our genes of interest in yeast cells. We will further use it to integrate our TRAPS system to target the mCherry RNA. The presence of a clear band at the expected size of ~5kb indicates a successful digestion. Bands are also visible at ~1kb and ~1.6kb confirming the correct size of the cut out fragment (combined size of ~2.6kb).
Figure 5: Isolation of Cas13-TRAPS Inserts
Figure 5. Agarose gel electrophoresis of Inserts and destination vector. (1 % agarose in 1× TAE buffer, stained with EtBr). Empty Vector pAG-416 cut with SacI and MluI, Insert Im2-MiniCas13.X1 and Insert GFP-E9-Tetra. 1kb Ladder is used as a size marker.
Figure 5 shows the successful isolation of our inserts Im2-MiniCas13.X1 and GFP-E9-Tetra after restriction digestion and gel extraction. The presence of clear bands at the expected sizes of ~3.2 kb for Im2-MiniCas13.X1 and ~1.2 kb for GFP-E9-Tetra indicates that our cloning strategy worked. The empty vector pAG-416 linearized with Mlu1 shows a band at the expected size of ~5 kb (+bands at the expected combined size (~2.6kb) of the cut out fragments), confirming that the vector is ready for ligation with our inserts. These inserts were then ligated together into the pAG-416 vector to create our TRAPS system.
Figure 6: Isolation of individual gRNAs
Figure 6. Agarose gel electrophoresis of gRNA inserts isolated. Gel was run after gRNA inserts were cut with gRNA1: SacI and EcorI, gRNA2: EcoRI and SphI, gRNA3: SphI and NotI and gRNA4: NotI and MluI to create compatible ends for ligation. (1 % agarose in 1× TAE buffer, stained with EtBr). 1kb Ladder is used as a size marker.
Figure 6 shows the successful cutting and isolation of our gRNA inserts at the size of ~250b. These inserts were then isolated via gel extraction and ligated into the pAG-305 vector to create our gRNA collection for our TRAPS system.
Figure 7: Isolation of Pumby-TRAPS plasmids
Figure 7. Agarose gel electrophoresis of Pumby-Plasmids after ligation of the inserts into the vector and restriction digestion of the vector for linearization with MluI. (1 % agarose in 1× TAE buffer, stained with EtBr). pAG416 Vector with pumby1 insert and pAG305 Vector with pumby 2 insert1kb Ladder is used as a size marker.
Figure 7 shows the successful isolation of our Pumby inserts cloned into the respective vectors after restriction digestion. The presence of clear bands at the expected sizes of ~8kb for pAG416-Pumby1 and ~10kb for Pumby2 indicates that our cloning strategy worked. In the pAG416-pumby1 samples is another band visible at ~0.5kb. This is because the vector was cut with Sac1 which cuts twice in the vector. The combined size of the drop out and the main band corresponds to the expected size of the vector of ~8.5kb thereby confirming that the vectors are ready for transformation into our yeast strain. The pAG305 is thereby a genomically integrating vector to ensure the yeast strain does not lose the plasmid. The pAG416 is a high-copy plasmid that allows for strong expression of our genes of interest in yeast cells.
Figure 8: Second version of Cas13-TRAPS Inserts
Figure 8. Agarose gel electrophoresis of the second version of Cas13-TRAPS Inserts cut with MluI/SacI and SphI. (1 % agarose in 1× TAE buffer, stained with EtBr). Insert Im2-RfxCas13d cut with Mlu1 and Sph1, and Insert GFP-E9-Tetra cut with Sph1 and Sac1. 1kb Ladder is used as a size marker.
Figure 8 shows the successful isolation of our second version of Cas13-TRAPS insert Im2-RfxCas13d and the previously used insert GFP-E9-Tetra after restriction digestion. The presence of clear bands at the expected sizes of ~4 kb for Im2-RfxCas13d (marked with red arrow) and ~2 kb for GFP-E9-Tetra show the successful isolation of our inserts. These inserts were then ligated together into the pAG-416 vector to create our updated TRAPS system with the RfxCas13d variant. Besides the band of the empty vector, there are more bands visible at different sizes. This could be due to incomplete cutting of the vector or binding activity of the enzymes or of the inserts themselves. After this gel electrophoresis a gel extraction of the main band was performed to ensure that only the correct band is used for the ligation. The ligation and transformation of E. coli showed successfully transformed colonies which were further verified via plasmid mini prep and sequencing.
Microscopy Images
Figure 9: mCherry expressing yeast colonies (BsuI)
Figure 9. Microscopy of gal-induced an uninduced yeast colonies after transformation with the genomically integrating plasmid linearized with BsuI. Total of four different colony microscopies are displayed.
Figure 10: Total cell fluorescence distributions (BsuI)
Figure 10. Histograms showing the distribution of total cell fluorescence in uninduced and gal-induced yeast cells transformed with the BsuI linearized plasmid.
Figure 11: mCherry expressing yeast colonies (MfeI)
Figure 11. Microscopy of gal-induced an uninduced yeast colonies after transformation with the genomically integrating plasmid linearized with BsuI. Total of four different colony microscopies are displayed.
Figure 12: mCherry expressing yeast colonies (MfeI)
Figure 12. Microscopy of gal-induced an uninduced yeast colonies after transformation with the genomically integrating plasmid linearized with BsuI. Total of four different colony microscopies are displayed.
As described above, we linearized the mCherry-containing plasmid and transformed the linearized plasmid into the W303 S. cerevisiae strain. After transformation, a set of the resulting colonies was imaged and a candidate for further experimentation was selected. Linearization with both BsuI and MfeI restriction enzymes resulted in successful transformation for most of the colonies, as indicated by the homogeneous fluorescence when galactose medium was used for cell growth (Figure 9,11). In addition, we plotted histograms of total cell fluorescence in uninduced and gal-induced yeast cells (Figure 10, 12) using the image analysis code (see Software), further showing ymCherry expression upon gal induction. For further experiments with our TRAPS constructs, a colony showing strong fluorescence from the transformation linearized with MfeI was selected.
Figure 13: Microscopy of TRAPS-Cas13 (dCas13X.1)
Figure 13. Microscopy of the first TRAPS-Cas13 iteration containing miniaturized dCas13X.1. A-C: TRAPS-Cas13 with gRNA targeting mCherry grown in glucose medium. D-F: TRAPS-Cas13 with gRNA targeting mCherry grown in galactose medium. G-I: TRAPS-Cas13 without gRNA targeting mCherry grown in glucose medium. J-L: TRAPS-Cas13 without gRNA targeting mCherry grown in galactose medium. M-O: Control with yeast cells containing the GFP without proteins fused. P-R: Control of WT yeast
The first version of the TRAPS-Cas13 proteins with the miniaturized dCas13x.1 variant was transformed and introduced into the yeast strain containing the galactose-dependent mCherry. Green droplets within the cells were visible with and without galactose induction, as well as with and without gRNAs (Figure 13C, F, I, L), indicating that droplet formation is independent of RNA’s presence. Droplet formation was not observed in the yeast strain where only GFP, without any fusion, was transformed.
Figure 14: Microscopy of TRAPS-Cas13 in WT yeast
Figure 14. Microscopy of TRAPS-Cas13 transformed into WT yeast and controls. A-C: TRAPS-Cas13 transformed into WT yeast. D-F: Control with yeast cells containing the GFP without proteins fused. G-I: Control of WT yeast.
To see if the droplet formation is completely independent of the presence of the target RNA, we transformed our constructs into W303 WT yeast, which do not contain the gene for mCherry. Even when transformed into yeast lacking the mCherry gene, droplet formation still occurred (Figure 14C), showing that no mCherry RNA is needed for this, further strengthening the hypothesis that our proteins aggregate
Figure 15: Microscopy of TRAPS-Cas13 tetramerization domain
Figure 15. Microscopy of the TRAPS-Cas13 tetramerization domain transformed into yeast and controls. A-C: TRAPS-Cas13 tetramerization unit transformed into WT yeast. D-F: Control with yeast cells containing the GFP without proteins fused. G-I: Control of WT yeast.
To determine which of the two proteins introduced into the cell aggregates, we separated the cassettes, added the respective missing restriction sites, and transformed the tetramerization unit individually. When only the tetramerization unit was transformed, we observed distributed cytosolic GFP fluorescence (Figure 15C) without droplets, indicating that the IM2-Cas13 unit is the driver of aggregation.
Figure 16: Second version of TRAPS-Cas13 with RfxCas13d
Figure 16. Microscopy of the second version of TRAPS-Cas13 containing the RfxCas13d variant. A-C: TRAPS-Cas13 with gRNA targeting mCherry grown in glucose medium. D-F: TRAPS-Cas13 with gRNA targeting mCherry grown in galactose medium. G-I: TRAPS-Cas13 without gRNA targeting mCherry grown in glucose medium. J-L: TRAPS-Cas13 without gRNA targeting mCherry grown in galactose medium. M-O: Control with yeast cells containing the GFP without proteins fused. P-R: Control of WT yeast.
After determining that the Cas13 unit is the driver of aggregation, we integrated a second version of the IM2-Cas13 protein using an RfxCas13d variant. We again observed droplet formation independent of the presence of our target RNA (Figure 16C, F, I, L). Similar aggregates formed as in the first TRAPS-Cas13 iteration. We realized that N-terminal fusion of this specific Cas13 version leads to aggregation (Han et al., 2020) and therefore designed a third iteration of the TRAPS-Cas13 system, which is currently in progress but has not yet been tested.
Figure 17: Microscopy of TRAPS-Pumby System
Figure 17. Microscopy of the TRAPS-Pumby System.1. A-C: TRAPS-Pumby targeting mCherry grown in glucose medium. D-F: TRAPS-Pumby targeting mCherry grown in galactose medium. M-O: Control with yeast cells containing the GFP without proteins fused. P-R: Control of WT yeast.
We also tested the TRAPS-Pumby system. Similar to the TRAPS-Cas13 system, droplets were observed independent of the target RNA (Figure 17C, F). The cause of this droplet formation is likely, again, aggregation of the Pumby domains or general instability of the fusion proteins, although this was not experimentally proven.
Protein Analysis
Figure 18: SDS-Page gel with GFP-signal from GFP-E9-Tetra protein constructs in yeast.
Figure 18. Lysates were prepared from the yeast cell lines W303 WT (WT), W303 mCherry Cas13 gRNA(constructs +gRNA) and W303 mCherry Cas13 (constructs -gRNA). 12% SDS Page gel after running 40 min constant 200 V. Molecular weight protein marker VI (10 - 245) prestained was used. First four lanes show cell lysate from W303 mCherry Cas13 gRNA: firstly the Total (T) cell lysate, first a not-heated, then heated sample. Secondly Supernatant (S) fraction, first not heated, then heated sample. Next four samples show cell lysate from W303 mCherry Cas13: T, fist not-heated, then heated sample, S non-heated, then heated sample. Lastly WT yeast cell lysate as control. GFP-Fluorescence signal at ~45 kDa can be seen in non-heated samples of W303 mCherry and W303 mCherry Cas13.
Lysates were prepared from the yeast cell lines W303 WT (WT), W303 mCherry Cas13 gRNA(constructs +gRNA), where gRNA is expressed alongside the constructs of interest and W303 mCherry Cas13 (constructs -gRNA), where no gRNA is expressed. Equal amounts of total protein were loaded for all lanes. Molecular weight protein marker VI (10 - 245) prestained was used. The first four lanes correspond to lysates from W303 mCherry Cas13 gRNA. Total cell lysate (T) was loaded first, with a non-heated (N) and then a heated (H) sample, followed by the supernatant fraction (S), also as a non-heated and heated sample. The next four lanes show lysates from W303 mCherry Cas13, in which the constructs are expressed without gRNA, again displaying total lysate and supernatant fractions in non-heated and heated conditions. The final lane contains lysate from WT yeast as negative control. A 12 % SDS Page gel was run for 40 min at constant 200 V. Afterwards gel was imaged in Amersham Typhoon. Distinct fluorescence bands can be detected in non-heated samples of W303 mCherry Cas13 gRNA and W303 mCherry Cas13 at the size of ~ 45 kDa (Figure 18). Next to the expected lanes a weak signal can be seen at ~27 kDa in TN and SN of mCherry Cas13. Afterwards the proteins from the gel were transferred onto a nitrocellulose membrane
Figure 19: Expression of Cas-Im2 and GFP-E9-Tetra protein constructs in yeast.
Figure 19. Lysates were prepared from the yeast cell lines W303 WT (WT), W303 mCherry Cas13 gRNA(constructs +gRNA) and W303 mCherry Cas13 (constructs -gRNA). Molecular weight protein marker VI (10 - 245) prestained was used. A Nitrocellulose membrane was probed with primary anti-Myc-Antibody (1:5000 in 2% skim milk, mouse) against Myc-tagged constructs Im2-Cas13 and GFP-E9-Tetra and secondary antibody Rabbit Anti-Mouse Alexa Fluor 488 (1:10000 in 5% skim milk, rabbit, fluorescence detection) was used. B Nitrocellulose membrane was probed with primary anti-PGK1-Antibody (1:5000 in 2% skim milk, mouse) for detection of housekeeping gene PGK1 (loading control) and secondary antibody Rabbit Anti-Mouse Alexa Fluor 790 (1:10000 in 5% skim milk, rabbit, fluorescence detection) was used.
The image presented was generated by probing the nitrocellulose membrane with anti-Myc-Antibody (1:5000 in 2% skim milk, mouse) for the detection of the constructs Im2-Cas13 and GFP-E9-Tetra and anti-PGK1-Antibody (1:5000 in 2% skim milk, mouse) for detection of housekeeping gene PGK1. The latter served as loading control to normalize the relative expression of the protein constructs between lanes. As secondary antibody Rabbit Anti-Mouse Alexa Fluor 488 (1:10000 in 5% skim milk, rabbit) was used for fluorescence detection of proteins of interest and Rabbit Anti-Mouse Alexa Fluor 790 (1:10000 in 5% skim milk, rabbit) for detection of PGK1. Distinct bands corresponding to GFP-E9-Tetra (~48 kDa) and Cas13-Im2 (~65 kDa) were observed in the TN and TH samples of gRNA-expressing cell lines (Figure 19A). In the absence of gRNA, both constructs were detectable across all fractions, including TN, TH, SN, and SH. Fluorescence of the loading control PGK1 was detected in all lanes (Figure 19B).
Figure 20: Quantification of protein expression.
Figure 20. Densitometric analysis was performed using the Gel Analysis tool in Fiji (doi:10.1038/nmeth.2019) on the image in Figure 2. Quantification revealed higher band intensity in cell lines without gRNA compared to cell lines with gRNA expression, higher band intensity in T compared to band intensity in S, higher band intensity of GFP-E9-Tetra in heated samples compared to Cas13-Im2 band intensity.
Densitometric analysis was performed using the Gel Analysis tool in Fiji (doi:10.1038/nmeth.2019). To correct for differences in sample loading, measured band intensity values were normalized to the corresponding loading control PGK1. Since band intensities in gRNA-expressing cell line W303 mCherry Cas13 gRNA did not exceed background levels for both SH and SN fractions, only TH and TN samples were used in analysis for gRNA expressing cell line. In the cell line W303 mCherry Cas13, without gRNA expression all samples (TH, TN, SH, SN) produced a measurable band signal.
| Sample | Condition | Cas13-Im2 signal | GFP-E9-Tetra signal | PGK1 signal |
|---|---|---|---|---|
| W303 mCherry Cas13 gRNA | TN | 1.466,426 | 1.176,820 | 10.511,095 |
| TH | 3.452,004 | 6.299,075 | 17.148,238 | |
| SN | - | - | 11.299,288 | |
| SH | - | - | 14.743,359 | |
| W303 mCherry Cas13 | TN | 4.868,660 | 7.365,782 | 16.956,995 |
| TH | 5.447,125 | 16.718,652 | 17.547,459 | |
| SN | 2.206,891 | 1.868,477 | 16.412,752 | |
| SH | 1.156,527 | 1.523,305 | 18.744,652 | |
| WT | TH | - | - | 19.287,116 |
Table 1. Comparison cell lines with gRNA vs. without gRNA:
Corresponding samples in both cell lines were compared and analysis revealed a constantly higher signal in in cells without gRNA compared to cell lysate with gRNA. A 2-fold higher TN GFP-E9-Tetra band intensity a 3.8 fold higher TN Cas13-Im2 band intensity, a 1.5 fold higher TH GFP-E9-Tetra and a 2,6 fold higher TH Cas13-Im2 band intensity in cell lines without gRNA expression in comparison to cell lines with gRNA expression was measured.
2. Comparison of Total (T) vs. Supernatant (S) fraction:
Corresponding samples in both cell lines were compared, as no band intensity higher than the background signal was measured in cell lines with gRNA expression for both SH and SN, only cell lines without gRNA expression were compared. For GFP-E9-Tetra samples a 3.8 fold higher band intensity was measured in TN than in SN and a 11.7 fold higher band intensity was measured in TH than in SH. For Cas13-Im2 a 2.1times higher band intensity was measured in TN than in SN and a 5 fold higher band intensity was measured in TH than in SH.
3. Comparison of Cas13-Im2 vs GFP-E9-Tetra expression:
The band intensity for the relative expression of the two constructs in each sample was analysed and signal intensity was higher for the GFP-E9-Tetra construct in comparison to the Cas13-Im2 construct in cell lines with gRNA for TH by a factor of 1.8, and for cell lines without gRNA in TN by a factor of 1.5, for TH by a factor of 3.1, for SH by a factor of 1.3. In the other samples the signal intensity was higher in Cas13-Im2 than GFP-E9-Tetra in by 1.2 in TN (with gRNA) and in SN (without gRNA).
Figure 21: SDS-Page gel with GFP-E9-Tetra protein constructs in yeast
Figure 21. Lysates were prepared from the yeast cell lines W303 WT (WT), W303 mCherry Cas13. 12% SDS Page gel after running 40 min constant 200 V. Molecular weight protein marker VI (10 - 245) prestained was used. Firstly WT yeast cell lysate as control. Next four lanes show cell lysate from W303 mCherry Cas13: firstly the Total (T) cell lysate, first a heated, then non-heated sample. Secondly Supernatant (S) fraction, with first heated and then non-heated sample. GFP-Fluorescence signal at ~65 kDa can be seen in non-heated samples of W303 mCherry.
Lysates were prepared from the yeast cell lines W303 WT, W303 mCherry Cas13. Equal amounts of total protein were loaded for all lanes. Molecular weight protein marker VI (10 - 245) prestained was used. The first lane contains lysate from WT cells, serving as a negative control. Next four lanes show lysates from W303 mCherry Cas13: first, the total (T) fraction, with non-heated (N) and heated (H) samples applied, followed by the soluble (S) fraction, also with non-heated and heated samples. A 12 % SDS Page gel was run for 40 min at constant 200 V. Afterwards gel was imaged in Amersham Typhoon. Distinct GFP fluorescence bands can be detected in non-heated samples of T and S at approximated molecular weight of 65 kDa and no other bands were observed (Figure 21). Afterwards the proteins from the gel were transferred onto a nitrocellulose membrane.
Figure 22: Western Blot of new Cas13 constructs.
Figure 22. Molecular weight protein marker VI (10 - 245) prestained was used. A) Nitrocellulose membrane was probed with primary anti-Myc-Antibody (1:5000 in 2% skim milk, mouse) against Myc-tagged constructs Im2-Cas13 and GFP-E9-Tetra and in B) anti-PGK1-Antibody (1:5000 in 2% skim milk, mouse) for detection of housekeeping gene PGK1 (loading control). Secondary antibody Rabbit Anti-Mouse Alexa Fluor 488 (1:10000 in 5% skim milk, rabbit, fluorescence detection) was used. Lanes: WT control, followed by total (T) and soluble (S) fractions, with heated (H) and non-heated (N) samples as indicated.
Western blot analysis was performed to assess the expression of Myc-tagged protein constructs in yeast cell lysates. Proteins were transferred to a nitrocellulose membrane and probed with anti-Myc antibody (1:5000 in 2% skim milk, mouse) to detect the constructs and anti-PGK1 antibody (1:5000 in 2% skim milk, mouse) for the housekeeping protein pGK1, which served as a loading control. Detection was performed using Rabbit anti-Mouse Alexa Fluor 488 (1:10000 in 5% skim milk) for fluorescence imaging. Molecular weight reference was provided by Protein Marker VI (10–245 kDa, prestained). Distinct fluorescence bands can be detected in TH and TN samples at approximated size of 65 kDa (Figure 22, top, middle two lanes). No second set of bands and no fluorescent signal were observed in the solube samples (Figure 22, top, outer right lanes). Fluorescence of the loading control pGK1 was detected in all lanes (Figure 22, bottom).
Figure 23: SDS-Page gel with GFP-PUM2-Tetra and GFP-PUM3-Tetra protein constructs in yeast.
Figure 23. Lysates were prepared from the yeast cell lines W303-mCherry-416-Pumb2y. 12% SDS Page gel after running 40 min constant 200 V. Molecular weight Protein Marker VI (10–245 kDa, prestained) is shown in first lane. Following lane shows SN followed by TN. Two distinct fluorescent bands at ~65 kDa and ~67 kDa and lighter signal at ~37kDa, ~39 kDa and ~25 kDa.
Lysates were derived from yeast cell lines W303-mCherry-416-Pumb2y, expressing the constructs of interest. Equal amounts of protein were loaded in all lanes. Molecular weight protein marker VI (10 - 245) prestained was used. The first lane after molecular weight marker contains non-heated Supernatant fraction from W303-mCherry-416-Pumb2y lysate, afterwards the non-heated Total fraction of W303-mCherry-416-Pumb2y lysate is applied. A 12 % SDS Page gel was run for 40 min at constant 200 V. Afterwards gel was imaged in Amersham Typhoon. Distinct GFP fluorescence bands can be detected in samples of T and S at approximated size of ~65 kDa and ~67 kDa. A lighter and less distinct signal can also be spotted in both T and S at ~37kDa, ~39 kDa and ~25 kDa. Afterwards the proteins from the gel were transferred onto a nitrocellulose membrane.
Figure 24: Failed western blot of Pumby constructs from yeast.
Figure 24. Nitrocellulose membrane was probed with primary anti-Myc-Antibody (1:5000 in 2% skim milk, mouse) against Myc-tagged constructs PUM1-Dim and PUM4-Tetra, anti-HA-Antibody (1:5000 in 2% skim milk, mouse) against HA-tagged GFP-PUM2-Tetra, anti-Flag-Antibody (1:5000 in 2% skim milk, mouse) against Flag-tagged GFP-PUM3-Tetra and anti-PGK1-Antibody (1:5000 in 2% skim milk, mouse) for detection of housekeeping gene PGK1 (loading control). Secondary antibody Rabbit Anti-Mouse Alexa Fluor 488 (1:10000 in 5% skim milk, rabbit, fluorescence detection) was used. Molecular weight protein marker VI (10 - 245) prestained was used. First two membranes were incubated with anti-Myc primary antibody. Second two membranes were incubated with anti-Flag primary antibody. Last two membranes were incubated with anti-HA-primary antibody. No signal from immunostaining is visible.
Western blot analysis was performed to assess the expression of Myc-tagged protein constructs PUM1-Dim and PUM4-Tetra, Flag-tagged protein construct GFP-PUM3-Tetra and HA-tagged protein construct GFP-PUM2-Tetra in yeast cell lysates. Proteins were transferred to a nitrocellulose membrane and probed with anti-Myc antibody (1:5000 in 2% skim milk, mouse) to detect Myc-tagged constructs PUM1-Dim and PUM4-Tetra, with anti-HA-Antibody (1:5000 in 2% skim milk, mouse) to detect HA-tagged GFP-PUM2-Tetra and anti-Flag-Antibody (1:5000 in 2% skim milk, mouse) to detect Flag-tagged GFP-PUM3-Tetraand. Anti-PGK1 antibody (1:5000 in 2% skim milk, mouse) was used for detection of the housekeeping protein pGK1, which served as a loading control. Detection was performed using secondary antibody Rabbit anti-Mouse Alexa Fluor 488 (1:10000 in 5% skim milk) for fluorescence imaging. Molecular weight reference was provided by Protein Marker VI (10–245 kDa, prestained). Membranes were cut into pieces, so that each membrane could be incubated into a different primary antibody. First two membranes were incubated in anti-Myc-primary antibody: the first Myc-incubated membrane contains non-heated samples, second membrane contains heated samples, each first Total (T) fraction then Supernatant (S). Second two membranes were incubated in anti-HA-antibody: first HA-incubated membrane shows non-heated samples, second membrane shows heated samples, each first T then S. Third two membranes were incubated in anti-Flag-antibody: first Flag-incubated membrane shows non-heated samples, second membrane shows heated samples, each first T then S. There was no immunostaining signal (Figure 24A) and fluorescence of the loading control pGK1 was detected in all lanes (Figure 24B).
References
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