I am currently trying to investigate an integral membrane protein expressed in Arabidopsis with a GFP tag. Using anti-GFP magnetic beads I am able to purify this protein. I would like to know the best way to elute my protein from the magnetic columns I use, while preserving any native complexes it forms (for downstream Blue Native PAGE analysis). I've read of a few ways to elute protein complexes, these include low pH shifts using 0.1M glycine-HCL. Using high pH shifts with 0.1M triethylamine, pH 11.8 with 0.1% Triton X-100 (not the detergent I started with to solubilise membranes). OR by removing the column from the magnetic field and eluting the beads with my protein of interest.... (this method produced large smears on my 1D BN-PAGE gel).
As it is with membrane proteins, its makes it more difficult to continuously provide a stable environment for complexes to stay intact, so any advice on specifically eluting membrane protein complexes would be of great help.
Many thanks for any suggestions.
If you can manage it, a good way to separate the immunoaffinity purified protein from the antibody under gentle physiological conditions is to compete the protein with the epitope of the antibody. This can be done using a synthetic peptide containing the epitope, assuming it is a continuous epitope and the epitope is known, but not a structural epitope. It could also be done, in your case, using pure GFP protein. Obviously, this method is expensive and has special requirements that the other methods do not have. Purified GFP is available commercially.
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