30 A Practical Guide to Cancer Systems Biology
- Centrifuge the lysate at 17,000 g, 4◦C for 30 minutes. Transfer the
supernatant, which contain the crude extracted proteins, to a new 1.5 mL
centrifuge tube. The protein sample can be stored at− 30 ◦C for 2–3 days
or stored at− 80 ◦Cformonths.
Reduction and alkylation of proteins^17
- Freshly prepare 200 mM TCEP and keep it at 4◦Cbeforestartingthe
following steps. - Measure the concentration of protein with Pierce BCA Protein Assay Kit.
- Transfer the amount of proteins to a new 2 mL protein lobind tube.
The amount of 10− 100 μg/sample is suitable for testing or small-scale
experiment, while 400μg/sample is enough for large-scale experiment. - Adjust each sample to have equivalent amount and volume with lysis
buffer. - Add 1 M TEAB to a final concentration of 50 mM TEAB in each sample.
- (Optional) Examine the pH with pH test strip. The pH of each sample
should be about pH 8.5. - Protein reduction by adding 200 mM TCEP to a final concentration of
5 mM TCEP in each sample. Incubate the sample at 37◦C on a heat block
for 30 minutes. - Protein alkylation b adding 200 mM MMTS to a final concentration of
2 mM MMTS in each sample. Incubate the sample at room temperature
for 30 minutes in the dark.
Gel-assisted digestion of proteins^17
- Add appropriate amount of 40% acrylamide/bis-acrylamide (37.5:1),
10% APS and TEMED sequentially. The volume of each reagent should
be in the following proportion:
Reagent Proportion in volume
Sample 14
40% Acrylamide/bis-acrylamide (37.5:1) 5
10% APS 0. 3
TEMED 0. 3- The step of adding TEMED must be done rapidly. Vortex and spin
down immediately after adding TEMED to sample. Let it stand at
room temperature for 10 minutes to allow gel polymerization.