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IMPACT system purification problem


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#1 BAK

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Posted 13 May 2010 - 12:30 PM

Hi everyone!

I have cloned a protein into a ptxb1 vector ( my protein is followed by the intein-chitin binding domain tag). The problem is that the construct appears to have different sized depending of using an antibody against my protein or against anti-chitn. Why is the reason of this shift in size if it is the same initial construct? Can two different antibodies recognized the same protein at different sizes if the protein has both epitopes.

The intein-chitin tag is about 27 Kda. The band detected using anti-chitin serum is not of that sized.

#2 Freemason

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Posted 17 May 2010 - 08:29 AM

Hi there,

what sizes do your bands correspond to in relation to the full-length protein? What size(s) do you observe on a Coomassie-stained gel? And what kind of samples do you load onto your blot (crude cell lysate, right out of the flask, or processed samples)?

Maybe your protein is degraded somehow during expression or processing, one part includes the antigenic region of your target-specific antibody, the other one includes the chitin binding domain.

Greetings,
Tom




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