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MAb detected on ELISA but no mIgG detected in WB

MAb ELISA Western Blot

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#1 antibody15

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Posted 24 June 2015 - 12:15 PM

I have purified antibody from hybridoma cell culture (grown with FBS in the media). When using these antibodies to coat plates, I can probe the plates with anti-mouse IgG and detect the coated antibody. However, if I run these same purified antibodies on western blot and probe with the anti-mouse IgG, I don't get any signal seen.

 

I see a heavy and light chain on SDS page but when I run a western blot the bands react with bovine IgG or IgM but not with mouse IgG

 

This same antibody was able to be used successfully in immunohistochem to light up the antigen in the tissue section.

 

Thoughts??



#2 Missle

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Posted 25 June 2015 - 10:58 AM

What do you mean "when I run a western blot the bands react with bovine IgG or IgM but not with mouse IgG"?  Are you probing your western with anti-bovine IgG?  To clarify, you are running the hybridoma purified antibody on the gel and transferring it to a membrane and probing with the anti-mouse IgG or you are transferring your antigen and probing it with your hybridoma purified antibody?







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