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detection of a protein band of protein complex

western blot multiple protein complex Laminils sample buffer primary antibody chemiluminescence

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#1 asaren

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Posted 09 May 2015 - 05:25 AM

If the protein A (60kda) and protein B (30kda) can form a protein complex together, is it possible to detect protein A band at 30kda (protein B location) when do western blot (incubating only anti-protein A)? Is SDS in Laminil's sample buffer sufficient to dissociate/solubilize all protein-protein interaction?

 

I have experienced a protein A detected at 30 kda but not sure whether it is really protein A, or left over protein band before stripping the membrane, or just another unspecific band.

 

Thank you very much



#2 JDSBlueDevl

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Posted 11 May 2015 - 07:52 PM

SDS is a very strong detergent that will dissociate all components of a complex.  This is actually what elutes immunocomplexes from your protein-linked agarose beads in an immunoprecipitation.  Therefore, there should be no Protein A at 30 kDa.  Actually, if your complex were bound together, there would be a supershift to 90 kDa.  If your antibody toward Protein A is detecting something at 30 kDa, that might be a nonspecific band.  You might want to check your blocking conditions.



#3 mdfenko

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Posted 12 May 2015 - 04:11 AM

also, sds in laemmli sample buffer is not sufficient to dissociate all protein interactions. some require reducing agent (eg 2-me, dtt) to break disulfide bonds (internal as well as external).


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#4 asaren

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Posted 12 May 2015 - 06:09 AM

 

also, sds in laemmli sample buffer is not sufficient to dissociate all protein interactions. some require reducing agent (eg 2-me, dtt) to break disulfide bonds (internal as well as external).

I added BME in Laminil buffer too

 

SDS is a very strong detergent that will dissociate all components of a complex.  This is actually what elutes immunocomplexes from your protein-linked agarose beads in an immunoprecipitation.  Therefore, there should be no Protein A at 30 kDa.  Actually, if your complex were bound together, there would be a supershift to 90 kDa.  If your antibody toward Protein A is detecting something at 30 kDa, that might be a nonspecific band.  You might want to check your blocking conditions.

Thank you JDS







Also tagged with one or more of these keywords: western blot, multiple protein complex, Laminils sample buffer, primary antibody, chemiluminescence

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