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SDS-page question


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3 replies to this topic

#1 yves

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Posted 06 May 2004 - 07:31 AM

I've have a purified protein sample. The protein is 25 kDa, but we always see another band at 50 kDa, nevertheless we used reducing conditions, SDS, Mercaptoethanol and we heated the sample. Any suggestions how a kind of a dimer of the protein can still exist after these manipultions?

#2 phdconsult

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Posted 06 May 2004 - 05:18 PM

The protein is demonstrating a hydrophobic aggregate tendency. You might see only 1 band in a 2-D gel. Some reports, vague though, have reported that increasing Ammonium persulfate concentration in the gel pre-mix breaks up these dimers. It all depends on the hydrophobic/glycosylation/modification content of your protein

#3 keithwu

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Posted 13 May 2004 - 04:03 AM

Hi

Do you have some reference about hydrophobic aggregate tendency? :(

#4 Shubenok

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Posted 25 May 2004 - 10:07 AM

Add b-mercaptoethanol or DTT to all SDS-buffers, or try to reduce disulphids and modify them by iodacetamid e.g., cause it can be disulphid-linked monomers.




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