Western blotting

Procedure

1. Cells were plated in standard growth medium to achieve approximate 80% confluency.
2. Wash the cells with ice-cold PBS twice and collect cells with RIPA lysis buffer. Immediately scrape the cells off the plate and transfer the extract to a microcentrifuge tube.
3. Sonicate the sample on ice (Uibra Cell, SONICS ); 3 cycle; Intensity, 45%; Time,
   ON: 15 sec, OFF: 15 sec), and centrifuge it at 13,000 rpm for 10 min at 4 °C.
4. Transfer supernatant to a clean microcentrifuge tube. Measure the protein concentration using the BCA Protein Assay Kit (Pierce TM).
5. Add 5×Loading Buffer to the supernatant. Heat the sample to 99 °C for 10 min.
6. Load 10-20 μg sample onto SDS-PAGE gel and load 5 μl Prestained Protein Marker (Thermo, 26616) to determine molecular weights.
7. Electrophoresis at constant75 Volts (about 30 minutes) usingrunning buffer until the protein dye reaches the separation gel and change to 120 Volts until the protein dye reaches the bottom of the gel.
8. Electrotransfer to PVDF membrane using transfer buffer (constant 80 Volts 1 h on ice).
9. Incubate membrane in 30 ml of blocking buffer (5% skim milk with TBST) for 2-3 h at RT. Wash three times for 5 min each with 20 ml of TBST.
10. Incubate membrane and primary antibodyin 10 ml primary antibodydilution buffer (TBST) with gentle agitation overnight at 4 °C. Wash three times for 10 min each with 20 ml of TBST.
11. Incubate membrane with HRP-conjugated goat anti-rabbit/mouse secondary antibody (1:5000-1:10000) in 10 ml of TBST with gentle agitation for 2 h at RT. Wash three times for 10 min each with 20 ml of TBST.
12. Incubate membrane with 4 ml BeyoECL Plus mix substrate (Beyotime, P0018) with gentle agitation for 2 min at RT.
13. Wrap the membrane in plastic wrap and expose to x-ray film. An initial 5-10 sec exposure should indicate the proper exposure time.
14. Scan the membrane. Calculate protein amount using Image J program.
    Note: Tanon Chemiluminescence gel imaging system was also used to obtain Real-time observation of chemiluminescence images.

Materials and Reagents

Solutions:

   1. 10×PBS (Phosphate buffer saline)

   Adjust pH to 7.4 by adding 5M NaOH.

   2. 10× TBS (Tris buffered saline)

   Adjust pH to 7.4 by adding hydrochloric acid.

   3. 1×TBST

   4. 5×Running buffer

   5. 5×Loading Buffer

   6. 10×Trans Buffer