Proteinase K Proteolysis Assay for S100 Proteins

Proteinase K proteolysis assay for S100 proteins

1. Exchange protein(s) into a useful buffer (see https://harmslab.uoregon.edu/wiki/harms-lab-protocols/protein-experiments/sample-prep-and-quality-assurance/). I usually use 25 mM tris, 100 mM NaCl, 1 mM TCEP – add 1mM EDTA or 1mM CaCl₂ for apo or calcium-loaded. You need at least 30 uL of 25 uM DIMER – measure concentration with Bradford, as A280 is unreliable for most of the calgranulins due to low extinction coefficients.
2. Pipette 30 uL of 25 uM dimer into a PCR tube – if testing more than one protein, use an 8-strip PCR tube.
3. In a separate 8-strip PCR tube, pipette 10 uL of 2X laemmli + BME SDS-PAGE gel loading buffer. You will need a tube for each time point tested. Label these tubes with your time points of interest (I typically do 0 mins, 1, 2, 5, and 30).
4. Set one thermocycler to 25 degrees C (or desired proteolysis temperature), and another to SDS boil (95 degrees C) to run for the duration of your experiment.
5. Place the laemmli-containing 8-strip tube in the SDS-boil thermocycler and put the caps on to prevent evaporation.
6. Remove 5 uL from each protein sample and pipette into the 0-min time point tube – if doing multiple proteins at a time, use the multi-channel pipettor. Add an additional 5 uL of buffer to each of your 0-min time point tubes. This is your no-protease control sample.
7. Make a 10 uM Proteinase K stock in buffer. For future use, I aliquot out 50 uL freezer stocks of PK so I only have to thaw it once – this helps with repeatability.
8. Make sure you’re all set up for the experiment and have a timer ready! Place your protein sample(s) in the 25 degrees C thermocycler, and then add 25 uL of 10 uM proteinase K to your sample(s) – use the multichannel P300 and the gray tips if you have more than one protein sample.
9. Roughly 10 seconds before a given time point, remove 10 uL from each sample using the multichannel pipettor. When your timer reaches a given time point, pipette the sample directly into its respective SDS-boiling tube to quench the proteolysis reaction. Keep caps on both your protein samples and SDS-boil samples when not doing this step to prevent evaporation.
10. You can remove SDS-boil tubes after 10 mins, but make sure they boil for a minimum of 10 minutes to inactivate the proteinase K.
11. Spin down your tubes and run the samples on a gel to visualize your proteolysis time course.