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SDS-PAGE Gel Electrophoresis Forum SDS-PAGE Forum and Gel Electrophoresis Forum. Discuss the running of agarose gels, sds-page gels, and other gels including sequencing, gradient or tricine.


Can't break dimers and anomalous migration

Can't break dimers and anomalous migration - SDS-PAGE Gel Electrophoresis Forum

Can't break dimers and anomalous migration - SDS-PAGE Forum and Gel Electrophoresis Forum. Discuss the running of agarose gels, sds-page gels, and other gels including sequencing, gradient or tricine.


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Old 04-04-2010, 04:10 PM
Pipette Filler
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Default Can't break dimers and anomalous migration



Hi guys, I have several funky things going on with my recombinant (His-tagged) protein. First it tends to aggregate and nothing I do seems to break it apart. A general procedure is heating the samples at 95 degrees C for 5-10 minutes, and I've used B-mercap, DTT, and TCEP to break disulfide bonds. The other issue is that it never seems to migrate according to its molecular mass (~15kDa for the monomer). Tricine gels have helped a bit but the worst part is the inconsistency...sometimes it lines up, sometimes not even close. I attached an image of the gel, the lanes closest to the ladder are what I'm frustrated about. The first 3 ladder bands are 15, 20, and 30 kDa respectively. The dimer lines up but the "monomer" is not even close. I know that both of these bands are my protein because I've done western blots and used His-stain to confirm it. Is it possible that the "monomer" is simply a dimer that is not totally denatured?
Thanks for reading!
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Old 04-04-2010, 04:27 PM
Pipette Filler
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Default Re: Can't break dimers and anomalous migration

Sorry, forgot to include the gel.
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anomalous , break , dimer , dimers , migration , recombinant , reducing


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