Western Blot

Add 4X Laemmli buffer to protein samples and incubate at 95^oC for 7 minutes.

Load samples into 12% polyacrylamide gel and run in running buffer at 80-100V for 60-120 minutes.

Transfer gel to 0.2μm nitrocellulose membrane at 350mA for 60 minutes at 4^oC.

Wash membrane with 1X TBS-T then block in 10% milk for 30 minutes at room temperature.

1X TBS-T: 10mM Tris-HCl + 150mM NaCl + 0.5% Tween-20 in H₂O, pH 8.0

Wash membrane with 1X TBS-T for 4x 5 minutes.

Incubate membrane in primary antibody solution overnight at 4^oC.

Antibodies diluted to the appropriate concentration in 2% bovine serum albumin in 1X TBS-T with 0.02% sodium azide

Wash membrane with 1X TBS-T for 4x 5 minutes.

Incubate membrane in secondary antibody solution for 1 hour at room temperature.

Secondary antibody (horseradish peroxidase conjugated) is diluted in 10% milk.

Wash membrane with 1X TBS-T for 4x 5 minutes.

Rinse ECL Clarity Solution over membrane 20x then image.

Once imaging is complete, wash membrane with 1X TBS-T x2 then H₂O x2 then let dry at room temperature overnight before storing.