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2d electrophoresis


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#1 pavani

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Posted 09 March 2010 - 06:01 AM

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 ,
Thank you

#2 mdfenko

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Posted 09 March 2010 - 07:40 AM

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,...)
talent does what it can
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#3 Inmost sun

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Posted 13 April 2010 - 07:57 AM

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

#4 bob1

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Posted 13 April 2010 - 04:23 PM

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.

#5 Inmost sun

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Posted 14 April 2010 - 12:32 AM

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.


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




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