Immunoblot sensitivity

Martin Blankfard Mblank at kpl.com
Mon Jul 24 14:10:22 EST 1995


In article <3uu1eh$j1q at news.doit.wisc.edu>,
   klenchin at macc.wisc.edu (Dima Klenchin) wrote:
>>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 

I would have to take issue with this statement.  HRP and TMB are at least as 
sensitive as AP and BCIP/NBT!!!  Our company produces both types of systems 
and I find the HRP/TMB to be much more sensitive and cheaper to use.




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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