Western Blot
ReagentVendorCatalog Number
RIPA buffer Life Technologies 89900
protease inhibitor cocktail Life Technologies 78440
BCA protein assay Life Technologies. 23227
NuPAGE® Novex® 4–12% Tris-Acetate Protein Gels Life Technologies NP0321BOX
PVDF membrane Millipore IPSN07852
SuperSignal™ West Femto Maximum Sensitivity Substrate Life Technologies 34094
Western Blot Analysis:
  1. To extract intracellular protein, cells were placed on ice, washed twice with ice-cold PBS and lysed immediately with RIPA buffer (Life Technologies, 89900) supplemented with a protease inhibitor cocktail (Life Technologies, 78440). Notes: Cell lysates need to be sonicated and the soluble fraction was collected after centrifugation.
  2. Protein amount was quantified by the BCA protein assay (Life Technologies, 23227).
  3. 20 μg of total protein for each sample was mixed with loading buffer and incubated for 5 min at 95 °C before being loaded into NuPAGE® Novex® 4–12% Tris-Acetate Protein Gels (Life Technologies, NP0321BOX) for electrophoresis.
  4. Separated proteins were transferred to a PVDF membrane (Millipore, IPSN07852) after electrophoresis. The membrane was blocked with 5% non-fat milk in TBST (50 mM Tris–HCl at pH 8.0, 150 mM NaCl, 0.1% Tween 20) followed by overnight incubation with the primary antibody at 4 ° C.
  5. A peroxidase conjugated horse anti-mouse IgG secondary antibody (1:20000, Vector Laboratories, PI-2000) or peroxidase conjugated goat antiRabbit IgG secondary antibody (1:20000, Vector Laboratories, PI-1000) was used for western blot.
  6. ECL signals were developed and detected using the SuperSignal™ West Femto Maximum Sensitivity Substrate (Life Technologies, 34094) and a CDiGit Chemiluminescent Western Blot Scanner (LI-COR).