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non specific immunoblot

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#1 ckp



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Posted 15 June 2015 - 04:42 AM

Developing the immunoblots on PVDF membrane  I realised a non-specific binding of the secondary antibody. It binds to my antigen when I miss the primary antibody. I want to check if my primary antibody binds to my antigen or not. Is it possible to stain the proteins (antigen alone and antigen with the primary antibody) on the PVDF membrane? With Commassie Blue? If somebody know it, please let me know..... Thanks!




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Posted 15 June 2015 - 05:39 AM

if you didnt use primary, you dnt know whether the secondary antibody is recognizing your protein of interest or something else around the same molecular weight.


you probably shd titrate down your secondary to avoid non-specific binding.  


alternatively, you can use the primary conjugated with HRP to detect your protein.

I always had an alternate hypothesis....

#3 mdfenko


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Posted 16 June 2015 - 03:57 AM

you can reversibly stain the membrane for protein with ponceau s. you can see a video of its use here (for some reason, this requires the internet explorer rendering engine to view, at least from firefox(i use ietab)).


and some info at this wikipedia page.

Edited by mdfenko, 16 June 2015 - 03:59 AM.

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