Western Blotting Protocol
Run SDS PAGE gel
Cut membrane to gel size (9cm x 6.5 cm) and soak in transfer buffer
Prepare sponge (4 pieces Whatman blotting paper). Cut to gel size and soak
in transfer buffer
Place gel in transfer buffer before transferring
On electrotransfer machine place 2 sponges, the membrane, the gel and
2 more sponges
-role out each sponge layer with a pipet to remove air bubbles
Run at 18V for 20 min
Block the membrane using 5% milk in 1 x TBS-tween for at least 1 hour (or overnight in cold room)
Add primary antibody to 5% milk- TBST in appropriate dilution for at least one hour (or overnight in cold room)
Wash in 1 x TBS- tween 5x's x 5 min or 3x's x 10 min
Add secondary antibody to 5% milk-TBST in appropriate dilution for about 1 hour
Wash in 1 x TBS-tween 5x's x 5 min or 3x's x 10 min
Add membrane to equal amounts ECL solutions 1 and 2 for 1 min
Wrap membrane in saran wrap
Place membrane in cassette and develop with film
Transfer Buffer (1 liter)
5.8g Tris base
2.9g Glycine
0.37g SDS
200mL Methanol
add H2O to 1L
10 x TBST
100mL of 1M Tris pH 8.0
20mL of Tween 20
300mL or 87.66g of 5M NaCl
Add H2O to 1L