[Protein-analysis] Re: IEF transfer
Dr Engelbert Buxbaum
engelbert_buxbaum at hotmail.com
Thu Jun 8 07:13:36 EST 2006
Brandon Hostetler wrote:
> To my point, I am trying to do an IEF slab gel and I have tried both
> nondenaturing and denaturing conditions in a pH range of 3-7. After I do my
> transfer I silver stain the gel and I have found a great deal of proteins
> still on the gel.
You will have to make sure that the proteins in your gel have bound
enough SDS first in order to give them a uniform negative charge.
Otherwise the proteins will not move out of the gel, as on the pI they
are uncharged and the buffereing capacity of the ampholytes may keep the
pH near the pI even in the presence of some sample buffer.
Incubate the gel with SDS-sample buffer first as you would do for
2D-electrophoresis. Then try either the tris/glycine buffer of Towbin et
al. or the Na2CO3/NaHCO3 buffer of Dunn.
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