General Western Blot Protocol*

Sample Preparation

1.	Remove media and wash cells with sterile PBS.
2.	Scrape cells into PBS and pellet in a centrifuge.
3.	Resuspend the pellet in 0.3 ml of RIPA buffer.
4.	Pass lysate through a 21-guage needle 3-4 times to shear the DNA.
5.	Incubate on ice for 30-60 minuutes.
6.	Spin at 15,000g for 20 munites at 4 degrees Celcius.
7.	Remove and siphon the supernatant (keep).
8.	Measure the protein content in the supernatant.
9.	Add loading buffer and B-mercaptoethanol or DTT to the supernatant.
10.	Boil samples for 3-5 minutes at 95 degrees Celcius (unless noted otherwise).

Western Analysis

1. Following transfer block the membrane for 1-2 hours in Block (5% NFDM, 1% BSA in TBS / 0.1% Tween).

2. Cut the blot into the appropriate strips and incubate each strip in a 15 ml conical tube with antibody diluted to the appropriate concentration in  5 – 10ml of Block. (Generally, antibodies are tested at 0.5 and 2.0 ug/ml initially.) Incubate 2 hours at R.T. or overnight at 4°C on a shaker.

3.  Rinse the blot once separately, then wash 6 x 5 minutes in TBS-Tween in a single 50 ml conical tube.  In the same tube wash 1x10 minute with diH₂O

4.  Dilute the appropriate secondary antibody in 25ml  Block and incubate 1 hour at R.T. or overnight at 4°C on a shaker. (Anti-Rabbit/anti-Mouse HRP is generally used at a 1:10,000 dilution.)

5.  Rinse the blot once, then wash 6 x 5 minutes in TBS-Tw followed by 1 x 20 minutes in diH₂O.

6.  Assemble blot on saran wrap or in a small staining dish and cover with ECL reagents made fresh according to the manufacturers instructions. Incubate for 10 minutes.

7.  Blot end of each strip on a paper towel to wick off ECL solution and assemble blot on saran wrap and seal. Be sure that the outside of the saran is completely dry as any moisture will interfere with the film processing. Be careful to never let blot become dry as this increases background greatly.

*The above information is only intended as a guide. The research should determine what protocol best meets their needs. Please follow safe laboratory procedures.