Western blot
Western blot is a method for analyzing a specific protein from a tissue or a cell extract. It was first tested by Dr. George Stark from Stanford and was named by W. Neal Burnette. In order to conduct a Western blot, the tertiary protein must be denatured into a primary structure for gel electrophoresis. Once the protein has been separated by size, it is transferred to a nitrocellulose or PVDF membrane for protein expression analysis using antigen-antibody interaction.

Western Blot Method
  • Separate the proteins of cell lysate via SDS-PAGE. (Refer to the cell lysis method below)
  • Place the SDS-PAGE gel in transfer buffer and transfer to a nitrocellulose (NC) membrane or a PVDF membrane.
  • Wash the transferred membrane with TBS-T (pH8.0) buffer for 10 minutes. Repeat 3 times.
  • Submerge the membrane in Ponceau S solution for 1-2 minutes in order to check for degree of transfer and bubbles.
  • Wash the membrane using D.W.
  • Submerge the membrane in a blocking solution (5% skim milk or 5% BSA) and leave on shaker for 1 hour.
  • Dilute the primary antibody with blocking solution and place on shaker for over 2 hours or overnight at 4℃.
  • Wash with TBS-T for 10 minutes. Repeat 3 times.
  • Dilute the conjugated secondary antibody that detects the primary antibody with a blocking solution and leave on shaker for over 1 hour.
  • Wash with TBS-T for 10 minutes. Repeat 3 times.
  • Express using West Save Up™(Abfrontier, Cat. No. LF-QC0101).

Coating buffer(500ml)
 50mM Tris(PH8.0)
2mM CaCl2
0.01% Antiform A
0.1% Tween-20
5% Skim milk
1X TBS-T(pH8.0)
 10mM Tris
0.15M NaCl
0.1% Tween 20

Cell Lysis Method

  • Wash the cultured cells with pre-chilled PBS 3 times and freeze at -70℃.
  • Pour lysis buffer in 100 π dish. (The volume of lysis buffer varies depending on the cell type and amount. Normally, 500µl per 1 sheet of 100 π is used.)
  • Harvest the cells in the tube using a scraper and let it sit in ice for 10 minutes.
  • Sonicate. Take caution to prevent the solution from heating.
  • Centrifuge at 4℃ for 20 minutes at 14,000 rpm.
  • Transfer the supernatant into a different tube and analyze.

Cell lysis buffer
 20mM Hepes(pH 7.2)
20mM Phosphoglycerate
0.15M NaCl
10% glycerol
1mM EDTA
1mM EGTA(Before use)
1mM PMSF
5μg/ml aprotinin & leupeptin
5μg/ml pepstatin A
1mM Na3VO4
5mM NaF