Solution Preparation
Lysis buffer:
0.15 M NaCl, 5 mM EDTA, pH 8.0, 1% Triton X100, 10 mM Tris-
Cl, pH 7.4. Just before use, add 5 mM DTT, 0.1 mM PMSF in
isopropanol, 5 mM ε-aminocaproic acid.
2X Sample buffer:
130 mM Tris-Cl, pH8.0, 20% (v/v) glycerol, 4.6% (w/v) SDS,
0.02% bromophenol blue, 2% DTT.
PBS, pH7.4:
10 mM Na2HPO4, 1.8 mM KH2PO4, 50 mM NaCl, 2.7 mM KCl
RIPA buffer:
50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Triton
X 100, 1% Na deoxycholate, 0.1% SDS, 1 mM PMSF, 1 µg/mL
aprotinin, 1 µg/mL leupeptin.
Protocol
A. Preparation of cell lysates (from T-25 flask)
1. Collect confluent cells by trypsinization and spin.
2. Lyse the pellet with 100 µl Lysis buffer on ice for 10 min
(use 20 µl Lysis buffer/500,000 cells).
3. Spin at 14,000 rpm (16,000 g) in a microcentrifuge tube for
10 min at 4°C.
4. Transfer the supernatant to a new tube and discard the
pellet.
5. Determine the protein concentration by Bradford assay.
6. Mix 1 volume of lysate (0.5 mg protein/membrane) with 1
volume of 2X sample buffer.
7. Boil for 5 min and cool at room temperature (RT) for 5 min.
8. Flash spin to bring down condensation prior to loading gel.
B. Preparation of tissue lysates
Always keep lysate or tissue on ice at all times during
preparation.
1. Remove tissues, and weigh 1.5 grams of each tissue.
2. Chop the tissue into small pieces, wash twice with ice-cold
PBS.
3. Transfer chopped tissue into grinder, and add 5 ml RIPA
buffer, homogenize 20 times.
4. Transfer homogenized solution into 1.5-ml microcentrifuge
tube. Spin at 14,000 rpm (16,000 g) for 10 min at 4°C.
5. Carefully remove the lipid on the surface of the
supernatant. Save supernatant as whole tissue lysate, and
discard the pellet.
6. Determine the protein concentration by Bradford assay.
7. Adjust concentration to 2.5 mg/ml with Lysis buffer. Aliquot
100 µl per vial, and store at -80°C.
8. For western blotting, add 100 µl 2X SDS sample buffer, boil
for 5 minutes, and store at -20°C.