How to reduce the dimers - (Oct/02/2007 )

I am using Ni-NTA beads to purify a protein. It turns out that that most protein form dimers after SDS PAGE analysis. So how to increase the proportion of the monomers? I sonicate the bacteria for half an hour. May be it's too long. Is there a relationship between the sonication time and the monomer/dimer formation? Does any one have any ideas? Thanks!

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some proteins naturally build dimers f.i. neurotrophic factors or growth factor receptors (phosphorylation dependent); in your case, it may be preparation-dependent aggregation;

sonication depends on not only on time but also on power; nevertheless, sonication for half an hour appears pretty long; what is the reason for such long duration as the risk of denaturation the protein and forming of aggregates increases?

you may get better resolution in SDS-PAGE using urea

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Hi, I am purifying the protein under denaturing conditions. So aggregation is not a big problem b/c I can use urea to dissolve them. I just don't want to get a lot of dimers b/c I need to analyze protein functions later. One thing I don't understand is that why the dimer is so strong? After boiling in SDS loading buffer, they are supposed to be totally denatured in my mind. So what is the interaction between monomers to form a dimer? disulfide bonds?

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disulfide bonds should be reduced by the Laemmli buffer; I still think in terms of aggregation of denaturized proteins; perhaps your protein tend to build dimers under native conditions, and dimeric aggregates are consolidated after denaturizing; may be the denaturized purification is not ideal for your protein...

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