Bio-Rad Immun-Blot® Opti-4CN™ Colorimetric Kits - Manual (Page 7)

2.3 Quick Guide
Blotting and Antibody Incubation
1. Transfer protein to nitrocellulose membrane by elec-
troblotting, dot blotting or microfiltration. Allow
membrane to air dry.
2. Wet membrane in PBST and then wash 2x for 5 minutes
each time in PBST.
3. Block membrane in 3% blocker for 1 hour.
4. Wash 2x with PBST for 3–5 minutes.
5. Incubate in appropriately diluted primary antibody
for one hour.
6. Wash 2x with PBST 5 minutes each time.
7. Incubate in 1:3,000–1:10,000 dilution of GAx-HRP *
secondary antibody for 1 hour.
8. Wash 2x with PBST 5 minutes each time.
Amplification (170-8238/39/40). All other kits,
skip to Detection.
9. Incubate membrane in diluted BAR for 10 minutes.
10. Wash 2–4x in 20% DMSO/PBST for 5 minutes each
time.
11. Wash 1–2x in PBST for 5 minutes each time.
12. Incubate membrane in diluted streptavidin-HRP for
30 minutes.
13. Wash 2x in PBST for 5 minutes each time.
9
3% Blocker. While vigorously stirring 665 ml of
PBST, very slowly add 20 g powdered blocker. Add the
powder a little bit at a time over a period of 30–45 minutes.
Continue stirring another 30–60 minutes after all the pow-
der has been added. Slowly warming the solution to 55 °C
will help put the blocker into solution, but do not overheat.
This can be accomplished by placing the flask with the
blocker solution inside a beaker on top of a magnetic stir-
rer/heater. Pour enough room-temperature water into the
beaker to surround the blocker solution as it stirs in the
flask. Be careful not to cause the flask with the blocker to
float or tip. Slowly heat the surounding water to 55–60 °C.
Do not add azide. Store at 4 °C. Warm to room temperature
before use.
Make these solutions on the day of the experiment.
Antibody dilution buffer (1% BSA in PBST).
Dissolve 0.75 g of BSA in 75 ml rapidly stirring PBST
(sufficient for 4 miniblots). Used for dilution of primary
and secondary antibodies and for dilution of streptavidin-
HRP.
Bio-Rad Amplification Reagent. Prepare 85 µl per cm 2
of membrane. Combine 2 parts 2x Amplification diluent,
1 part 4x BAR and 1 part ddH
2
O.
Streptavidin-HRP. Prepare 85 µl per cm 2 of mem-
brane. Dilute 1:1,000 with antibody dilution buffer.
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