For simplicity and wishing to avoid redundancy, our protocols section only includes protocols which are either unique to our project, or were improved by our team during the wet lab testing period. For an in-depth description of all of our experiments, please refer to the Lab Notebook section, which has both embedded links to standard protocols as well as complete written protocols.
Introduction
Protocols
Whilst other researchers have proposed the use of HiBit tagging to track the internalization of receptors, in our project the aim was slightly different: to track the internalisation of the protein itself. Therefore, through a series of experiments, we perfected and optimised a protocol for performing such measurement.
Materials
• BV-2 cells seeded in a 96-well plate at 70% confluence
• HiBiT-tagged protein of interest purified or in culture medium
• Nano-Glo® HiBiT Extracellular Detection System (Promega)
o LgBiT protein
o HiBiT Extracellular Substrate
o HiBiT Extracellular Buffer
• Multichannel pipette and reservoirs
Protocol steps:
1. Prepare protein stock
o Dilute the HiBiT-tagged protein in complete culture medium to the desired working concentration (based on sensitivity and resolution of plate reader) .
o Keep an aliquot aside for the t₀ reference.
2. Apply to cellso Remove medium from cells.
o Add 50 ul protein stock to each well of the 96-well plate containing BV-2 cells.
3. Timed medium removalo Remove supernatant sequentially every 3 minutes to capture uptake kinetics.
o Example: In our experiments, each row had a different timepoint, therefore, we would remove Row A at 3 min (vs. t₀), Row B at 6 min, Row C at 9 min, etc.
o When measuring 8 or more timepoints (without t0), use two or more plates in parallel:
Plate 1: t₀ to t₇ time points
Plate 2: t₀ (for normalisation) and t₈→ tn,
4. Prepare Extracellular Reagento Dilute LgBiT protein 1:100 and HiBiT substrate 1:50 into Extracellular Buffer (room temperature), so that the final detection mix’s volume is equal to the (n+4)*V, where n is the number of wells you are using, and V is equal to the volume of medium you remove from each well (or keep for t0)
Example: If you measure a total of 7 timepoints and a t0, with each time point in triplicate, and 4 different test groups at each timepoint, you would need a (96+4)*50 = 5mL total Extracellular Reagent.
o Mix thoroughly in a reservoir suitable for multichannel pipetting.
5. Add Extracellular Reagento Dispense into each well immediately after medium removal.
o Mix gently by pipetting up and down.
6. Incubateo Allow plate to sit for 15 minutes at room temperature in the dark after adding the detection mix.
7. Read signalo Measure luminescence using a plate reader.
o Normalise uptake across plates using t₀ controls
Notes:
• Sequential removal and immediate addition of detection reagent enables precise kinetic curves.
• Results are expressed as luminescence relative to t₀ to account for baseline protein exposure.
This protocol is adapted from the manufacturer’s SimpleStep GFP ELISA kit ( See more info). Our goal was to measure the GFP binding and internalising of our GFP-binding LYTAC variant directly in conditioned medium rather than in purified form. This implied several deviations from the manufacturer’s protocol, due to low concentrations (approximated through HiBiT luminescence measurements (0pg/mL to 0pg/mL7)) and incompatibility with the kit’s desired use (recommended use is cell extraction, tissue). Therefore, several steps were modified to account for the complexity of medium (secreted proteins, metabolites), to ensure valid comparisons between standards and samples.
Summary of adaptations versus standard protocol:
• Standards: prepared in 1:1 conditioned medium from pcDNA transfected cells and Cell Extraction Buffer instead of plain buffer, to ensure standards and samples share the same background.
• Sample prep: conditioned medium mixed 1:1 with Cell Extraction Buffer
• Standard curve: 2-fold dilutions starting from ~70 pg/mL down to 0 pg/mL, rather than the manufacturer’s starting concentration (20,000 pg/mL) were chosen because secreted protein levels were low.
• TMB development: extended beyond 20 minutes to achieve measurable color change, due to lower concentrations
Materials
• Conditioned medium samples (mixed 1:1 with Cell Extraction Buffer)
• Nano-Glo® SimpleStep GFP ELISA kit (Antibody Cocktail, Wash Buffer PT, TMB Development Solution, Stop Solution)
• GFP standard provided in kit
• Multichannel pipette, reservoirs, plate shaker, 96-well plate
Preparation of standards
Always prepare fresh; discard after
use.
1. Reconstitute GFP standard in 1× Cell Extraction Buffer PTR (per vial instructions) to make a 20,000 pg/mL stock.
2. Label eight tubes: Standards 1–8.
3. Add 376 µL conditioned medium + Extraction Buffer (same as samples) to tube 1, and 150 µL to tubes 2–8.
4. Make a 2-fold serial dilution from stock down to Standard 7; Standard 8 is blank.
o In our case, this yielded a 70 pg/mL → 0 pg/mL curve suitable for the concentration of our secreted proteins.
Protocol steps:
1. Equilibrate reagents, standards, and samples to room temperature.
2. Add 50 µL of each standard or sample per well (run in duplicate).
3. Add 50 µL Antibody Cocktail to each well.
4. Seal and incubate for 1 h at room temperature on a shaker (400 rpm).
5. Wash wells 3× with 350 µL Wash Buffer PT (≥10 s contact each wash). Invert and tap plate to remove liquid.
6. Add 100 µL TMB Development Solution to each well. Incubate in the dark on a shaker (5-20min).
o Adaptation: extended incubation to 30 min to develop sufficient signal.
7. Add 100 µL Stop Solution. Shake for 1 min.
8. Record OD at 450 nm (endpoint).
Notes
• Standards in conditioned medium ensure results reflect the same chemical environment as samples.
• Lower starting concentration (70 pg/mL) prevents wasted reagents and better matches expected sample levels, allowing for higher accuracy.
• Extended TMB incubation compensates for reduced color development in complex medium.
• All values normalised to the conditioned medium standard curve
In order to properly test our logic gates, we had to produce cells which had very limited background. It was decided that this would be done through switching from standard media (10% FBS) to starvation media (2% FBS). Thus, we transiently transfected HEK293 cells using PEI under low-serum (“starvation”) conditions, minimizing serum interference with DNA uptake.
Materials
• HEK293 cells (adherent)
• Culture media: DMEM (or appropriate basal), with and without serum
• Starvation medium: basal DMEM + %2 FBS
• Plasmid DNA, high purity, no contamination
• Linear PEI (e.g. 25 kDa), stock ~1 mg/mL, pH neutralized, sterile-filtered
• Opti-MEM or equivalent serum-free medium (for complexation)
• 1.5 mL Eppendorf tubes
• Pipettes, sterile tips
• 6-, 12-, 24-, 96- well plates (as needed)
• CO₂ incubator (37 °C, 5%)
• Sterile PBS (optional, for washes)
Before Transfection (Day 1)
1. Plate HEK293 cells so that they will reach ~60–80% confluence on the day of transfection.2. Use regular complete medium (DMEM + 10% FBS) until transfection.
Day of Transfection (Day 2)
1. Switch to starvation medium (if needed): ~1–2 h before transfection (or at the time of transfection), replace the culture medium with your starvation medium (2% FBS). This ensures that serum components don’t interfere with complex formation or uptake.
2. Prepare DNA solution:
o Calculate the total DNA needed per well (or per plate).
o Dilute DNA in Opti-MEM to a small volume (e.g. 50–100 µL) per well.
3. Prepare PEI solution:
o Use a typical DNA:PEI mass ratio of 1:2.5
o Dilute PEI in the same volume of Opti-MEM (or serum-free medium) as used for DNA (to the same final volume).
4. Complex formation:
o Add the PEI solution into the DNA solution, gently pipette up and down (avoid bubbles).
o Incubate at room temperature for ~10 min (up to 15 min, min 5 min) to allow complexes to form.
5. Add complexes to cells:
o Add the DNA–PEI complexes dropwise onto cells (e.g. along the side of the well) while gently swirling the plate to ensure even distribution.
o Avoid disturbing the monolayer.
6. Incubation:
o Incubate cells under standard conditions (37 °C, 5% CO₂).
o Leave the complexes on cells for 4–6 h to overnight, depending on toxicity and cell health.
7. Optional medium change:
o If toxicity is high, after 4–6 h you may gently replace the medium with fresh starvation medium. However, for tighter control, especially under stress conditions, you should not change the medium.
After Transfection (24–48 h)
1. Continue culture in starvation medium or switch back to normal complete medium (depending on your experimental design).
2. At 24–48 h post-transfection, assay for reporter expression (in our case nanoLuc) and cell viability.
3. Collect samples (lysates, supernatant) as required for downstream assays.