Protein purification - (Feb/20/2007 )

Hi , can anyone direct me to a protocol for E-GFP tagged protein purifications. I want to express these E-GFP proteins in mouse myoblast , extract them then purify them to use them later on in live cell imaging.


Cheers

Nancy

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if you overexpress a GFP-tagged protein, you be satisfied to enrich it by immunoprecipitation with anti-GFP Ab-beads;

if you are really interested in purification, which means actually pure i.d. absence of any other protein in silver stained gels, you may need several steps beside immunoaffinity enrichment by anti-GFP; other steps may include: cationic and anionic exchanger chromatography, gel filtration, chromatofocussing, hydrophobic interaction chromatography, other than GFP affinity chromatography f.i. your protein of interest; regard that you need a lot of myoblasts for purification

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Txs Bearer, i have thought about doing IP using E-GFP antibodies but then the purity and the yield is another issue as you mentioned before. It looks like purifying E-GFP tagged proteins is a bit tedious and requires optimistion , but i am conidering now just microinjecting directly my plasmids into the xenopos oocyte.

Cheers,

Nancy

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A good idea! Let's living cell do hard work itself!
Good Luck!

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