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recombinant protein lost immunological activity

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#1 scho



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Posted 23 March 2005 - 01:15 PM

I have a question about recombinant protein.
I got a recombinant protein using invitrogen TOPO system.
It is soluble protein and obtained high concentration of protein.
But it didn't react with monoclonal antibody when i did western blotting assay.
It could be lost their activity somehow. Actully i check DNA sequence, there is 99.2% of identity.
You guys have any comment welcome!!!

#2 jadefalcon



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Posted 29 March 2005 - 02:59 AM


Though solubilty is a good indicator for correct folding of your protein, there's no guarantee! Correct folding often is achieved by chaperons or the like in the original organism, when expressed recombinantly in E.coli, there can be problems such as yours. For your mAB: one possibility is, that the AB recognises a linear epitope that's due to different folding of the protein now inaccesible to the AB (cryptotope) - this could be ruled out by denaturing SDS-PAGE/Westernblotting, or your mAB recogises a structural epitope, which is destroyed due to incorrect folding, or your mAB recognises the section of your protein where the .8% difference occur (sometimes even a single change in one aminoacid in an linear epitope can abrogate AB-binding for good).
you could try to denaturate and refold your protein and see if there's some acitvity coming back. otherwise, I'd recommend you to check your AB, sometimes an aliquot is contaminated and therfore "eaten up"...

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