Bio-Rad Comparative Proteomics Kit II: Western Blot Module - Manual (Page 8)

Lesson 4 Quick Guide
1. If not blocked overnight, immerse
membrane in 25 ml blocking solution for
15 minutes to 2 hours at room tempera-
ture on a rocking platform.
2. Discard blocking solution and incubate
membrane with 10 ml of primary antibody
for 10–20 minutes on rocking platform set
to a faster setting to ensure constant
coverage of the membrane.
3. Quickly rinse the membrane in 50 ml of
wash buffer then discard the wash.
4. Add 50 ml of wash buffer to membrane for
3 minutes on rocking platform at a
medium speed setting.
5. Discard the wash and incubate membrane
with 10 ml of secondary antibody for
5–15 minutes on rocking platform set to
a fast setting.
6. Quickly rinse the membrane in 50 ml of
wash buffer and discard the wash.
7. Add 50 ml of wash buffer and wash
membrane for 3 minutes on rocking
platform on a medium speed setting.
8. Discard the wash and add 10 ml of HRP
color detection reagent.
9. Incubate 10–30 minutes, either with
manual shaking or on a rocking platform,
and watch the color development.
10. Rinse the membrane twice with distilled
water and blot dry with paper towel.
11. Air dry for 30 minutes to 1 hour and then
cover in plastic wrap or tape in lab book.
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Q
UICK GUIDE
Quick Guide