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Directly labled Antibody gives poor results

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#1 pkay



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Posted 23 August 2012 - 03:36 AM

hi everybody,

I am experiencing a weird problem. I have sucessfully prapared a recombinant human protein which is believed to bind to the surface of some human cells. When incubated with cells and subsequent detection I only get good results (i.e. Protein is detected on 90 % of the cells) when detection is indirect (same results with a-Protein+a-goat-PE and a-his+a-mouse PE). When incubated with a-Protein-PE antibody there are only about 2 to 5 % positive cells. On Cells expressing the protein I purified the antibody works.

In other Experiments like Western Blotting/IP I also get good results with my non-labled antibody.

Now I ask you whats the problem with the labled antibody in detecting my recombinant protein after binding to the cells. Does the PE-label inhibit binding?

Thank you in adavance!

Edit: Some further Information: Labled and non-labled Antibody are the same antibodies (same Manufacturer; same host; the differ only by PE-label); controls give no background staining by secondary antibodies!

Edited by pkay, 23 August 2012 - 03:51 AM.

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