Summary: Kate Shen 's Protocol Series: Western Blot
Protocol for Western Blot
(By using ECL Plus Western Blotting Detection Reagent from Amersham)
1. Electrophoresis and Blotting:
a. Prepare 10%SDS Page gel.
b. 1) If the samples are from tissue directly, dilute 25ug/7.5ul with your 7.5ul extraction
buffer + protease inhibitor (1:7 mix).(this volume is for mini gel with 15 well comb)
2) If the samples are from GST purified protein, 62.5ng are good amount of protein for
transfer and detection.( see bottom of my Western Blot picture)
c. Rinse the well with 1XSDS gel running buffer in tank, then loading the samples.
d. Run the gel at 180V for 45 minutes.
a. Cut off and trash the stacking gel.
b. Soak the resolving gel in 10mM CAPS/10% methanol buffer.
c. Soak the transfer membrane in methanol for 5 second, rinse it with dH2O, and then
equilibrate the membrane in transfer buffer for at least 15 minutes.
d. Assemble the blotting sandwich in the following order: black portion of cassette, sponge,
Whatman paper filter paper, PVDF membrane, Whatman paper filter paper, sponge,