Problems with NBT/BCIP visualization - (Mar/23/2008 )

I have encountered a problem when adding the NBT/BCIP substrate, my membrane got purple spots or stretches. How can i get rib of them?

---

perhaps insufficient blocking or washing

---

Make sure you spin down the antibodies before diluting into buffer. You may also want to dilute the antibody more. How are you blocking?

A good way to optimize this is to spot serial dilutions of your primary onto membranes, then block, detect, and wash with secondary. This lets you optimize for sensitivity and low background without the setup cost of doing westerns.

---

My conditions:
5% skim milk, block for 1hr
Primary Ab, 1:1000, 1 hr
Secondary Ab, 1:4000, 1hr

I just don't know the rationale of spinning down Ab, what will be the differences?

---

gets rid of particulates (including clumps of antibody with active phosphatase).

you may want to try more dilute secondary (10000x) and maybe also primary.

you should determine the optimum dilutions for both (can be done with dot or slot blots, as mentioned in an earlier post).

---