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-   -   Can we use dot blot inplace of Western blot? (http://www.molecularstation.com/forum/western-blot-forum/26413-can-we-use-dot-blot-inplace-western-blot.html)

peterish 11-01-2008 12:15 AM

Can we use dot blot inplace of Western blot?
 
You know dot blot is much easy to do than Western blotting, and in few occasions that we will lose the signal. Should we use dot blot to replace Western blotting?
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On the other hand, I wonder if dot blot can be used for samples eluted from immunoprecipitation. Your help and opinion is respected.

danfive 11-01-2008 03:13 AM

Re: Can we use dot blot inplace of Western blot?
 
Yes, dot blot is excellent. Especially if you can document that the Antibody detects your target protein in the Western, and the detection is specific----no nonspecific bands. So if you have a great Western blot set up for your target protein, go ahead and begin doing dot-blots.

peterish 11-01-2008 06:37 AM

Re: Can we use dot blot inplace of Western blot?
 
I got it. Thank you for your tips.

garengaston 06-16-2010 07:59 PM

Re: Can we use dot blot inplace of Western blot?
 
As for Dot-blotting IP eluant, you will get false positives from your Ab used in the IP.

Two ways around this:
1. Use a primary Ab for probing the membrane that is from a different organism than the Ab used for IP. (Obviously this is an expensive prospect.)

2. Below's the name to an interesting paper on using Protein A or G conjugated to HRP in place of secondary Ab. It ideally does not detect denatured Ab and will therefore only bind to the probe Ab. The site won't let me link to it so, here's the title "Clean western blot signals from immunoprecipitated samples" (A. Lal 2005)


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