2d electrophoresis - Substitution of Isoelectric focusing step in 2D-Electrophoresis ??? (Mar/09/2010 )
Now i would like to isolate and purify a perticular protein from breast cancer patients ,
but we don have 2D electrophoresis facility,
so wat im thinking is, instead of iso elecric focusing step in 2D-E, i would like to go for firstly " Ion exchange chormatography of the sample" then want to perform "SDS-PAGE..."
*Is this possible practically ??
*Is there any methods to isolate & purify a specific from the samples "with out using any Anybody Kits ??"
*Please mention by which method iso elecric focusing step substituted
Please kindly tell me ,
you could use chromatofocusing.
or you could use ion exchange with a pH gradient to elute.
simple ion exchange will not give the same selectivity as isoelectrofocusing.
you may be able to purify by affinity chromatography (substrate, inhibitor, cofactor,...)
I think the limitation of the biological material will be the problem; if you have an idea of the nature of the protein of interest, you may enrich it with an antibody, and then perform an 1D-GE
If you can identify the protein as a band on a normal 1D gel, you can cut it out and do a tryptic digest and then send it for identification by mass spectroscopy.
bob1 on Apr 14 2010, 12:23 AM said:
good idea but I recommend to loose some bulk proteins by enrichment of the protein of interest; more than 2 polypeptides in a cut-out band of an 1D gel is difficult to discriminate in MS