I would like to perform a modified co-immunoprecipitation assay using a GFP-tagged protein. We are going to bind the tagged protein to anti-GFP antibody then bind that to protein A/G beads, however we want to be able to selectively elute the tagged protein off the beads. I know that you can elute a FLAG tagged protein off of M2 anti-FLAG antibody by adding excess FLAG, so can I use the same approach with my GFP tag? If so, does anyone know a commercially available GFP monoclonal antibody who's epitope has been mapped so I can use excess epitope to elute my protein? Or possibly a source of recombinant GFP?
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I found a paper that describes a method to purify GFP fusion proteins using affinity chromatography: "Purification of GFP fusion proteins with high purity and yield by monoclonal antibody-coupled affinity column chromatography" (1). Unfortunately I don't have access to the full text, so I can't check how exactly they elute the GFP fusion protein from the column. They describe a specific monoclonal antibody they use, there should be information on how to obtain it in the paper.
Abstract:
GFP has often been used as a marker of gene expression, protein localization in living and fixed tissues as well as for protein targeting in intact cells and organisms. Monitoring foreign protein expression via GFP fusion is also very appealing for bioprocess applications. Many cells, including bacterial, fungal, plant, insect and mammalian cells, can express recombinant GFP (rGFP) efficiently. Several methods and procedures have been developed to purify the rGFP or recombinant proteins fused with GFP tag. However, most current GFP purification methods are limited by poor yields and low purity. In the current study, we developed an improved purification method, utilizing a FMU-GFP.5 monoclonal antibody (mAb) to GFP together with a mAb-coupled affinity chromatography column. The method resulted in a sample that was highly pure (more than 97% homogeneity) and had a sample yield of about 90%. Moreover, the GFP epitope permitted the isolation of almost all the active recombinant target proteins fused with GFP, directly and easily, from the crude cellular sources. Our data suggests this method is more efficient than any currently available method for purification of GFP protein.
answered Dec 16, 2011 at 13:27
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$\begingroup$ In the reported paper the authors used an affinity matrix carrying a covalently coupled mAb. They then eluted the bound proteins using the standard low pH glycine buffer. $\endgroup$ May 9, 2014 at 15:58