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recombinant protein has a bigger MW than control which is the same protein

recombinant protein has a bigger MW than control which is the same protein - Western Blot Forum

recombinant protein has a bigger MW than control which is the same protein - Discuss western blotting and immunoblot in the western blot forum discussion board. Ask Questions about transfers, blocking, membrane antibody incubation and exposures.


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  #1  
Old 02-22-2008, 07:21 PM
Pipette Filler
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Question recombinant protein has a bigger MW than control which is the same protein



I induced E.coli-cells with IPTG to express a protein which is 8-13 kDa big in human. the bands show a higher MW (between 15-20 kDa). the primary antibody bound on the control protein and on the bigger one from E.coli. Should it be that i have a wrong vector in my bacteria or is it a modification on the protein? Does anybody have an idea?
i will be happy if someone can answer.
bye, trypsin
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Old 02-26-2008, 01:03 PM
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Default Re: recombinant protein has a bigger MW than control which is the same protein

Hello there trypsin

it could be several things as you mentioned
1) cloning problem or addition of extra sequence into the protein (or new start site / or stop site)

2) addition of glycosylation residues on the protein (phosphorylation wouldnt increase it that big

3) ladder issues with your protein marker (maybe you mistakened it for the wrong site)

4) salt issues / buffer issues can easily change the estimated size of your protein

5) aggregation or binding to another protein? possibly due to bad lysis/lack of beta-mercaptoethanol in sample / lysis buffer.


I am out of ideas for more possibilities... anyone else?

please tell us what happened.
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Old 01-26-2009, 03:09 PM
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Default Re: recombinant protein has a bigger MW than control which is the same protein

Dimer?
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