Immunoblot sensitivity

Dima Klenchin klenchin at macc.wisc.edu
Sun Jul 23 12:38:25 EST 1995


>Does anibody know which are the inferior limit for detectability of a
>protein by silver staining and immunoblot or, at least which method is
>more sensitive?

It's a question that requires hours to answer in satisfactory detailes 
and review all alternatives. In brief: 

1. total protein with silver in gel - 1 ng should be easy; one rarely 
needs more in gels; in blots besides standard India Ink, you can go for 
colloidal gold (+ or - silver enhancer) or, if you biotinilate your 
total proteins before el.phoresis, you can go for enzyme detection - 
this will be most sensitive. 

2. Immunoblots. HRP is less sensitive than AP. Chemiluminiscence is more 
sens. than color development (other things being the same). 3 layers is 
more sensitive than 2 layers. ABC is more sensitive than conjugates. For 
extreme sensitivity I think the best is: 1. primary polyclonals, 2. 
secondary biotinilated (X- linker will work better) polyclonals, 3. ABC 
with AP, 4. chemiluminiscence. No need to say that (for extreme cases) 
all blocking steps and appropriate dilutions of reagents have to be 
optimized. 

Example: with BCIP/NBT and the above 3 layer procedure (ABC 
is homemade but available from Vector and Pierce) I have no problem 
detecting 1 pg of 23K protein on Western. The problem: I almost never 
need that much sensitivity :)) 

I will eventually compile a complete protocol for homemade ABC which is 
~ 10 times cheaper (with suppliers, dilutions, etc) if there will be 
enough requests. 

- Dima



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