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antibodies against membrane proteins


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3 replies to this topic

#1 ines stein

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Posted 02 November 2004 - 01:56 AM

we have problems with our antibdies against a membrane protein monoclonal and polyclonal antibodies are generated against the denaturated recombinant potein.
they work very well and specific in immunofluorescence, but do not work in western blotting analysis. what happend?

#2 Simonsays

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Posted 02 November 2004 - 01:45 PM

One of the solution would be to change your lysis buffer. If your buffer isn't strong enough to solubilize membrane proteins, your protein of interest stay in the lipid pellet after centrifugation. You may want to add Triton, SDS, deoxycholic acid or NP40. Check in the literature for the concentration, everybody gives their lysis buffer in the material and method section.

Hop I could help.

Simon

#3 wirly

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Posted 02 November 2004 - 03:15 PM

It is very common for an Ab to stain well in IF but not work at all on western and visa versa.

Granted, it's a bit strange (but not unheard of) that even in your POLYclonal you got no/poor staining on a western, but it's very well documented that every monoclonal antibody is unique in it's range of staining ability.

How many antobodies did you make?

#4 biomed

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Posted 18 November 2004 - 12:21 PM

One of the solution would be to change your lysis buffer. If your buffer isn't strong enough to solubilize membrane proteins, your protein of interest stay in the lipid pellet after centrifugation. You may want to add Triton, SDS, deoxycholic acid or NP40. Check in the literature for the concentration, everybody gives their lysis buffer in the material and method section.

Hop I could help.

Simon

Hi, Simon,
Definitely I can not agree you more. Lysis buffer is very critical for protein analysis.
Biomed
www.bmp-us.net




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