- 帖子:459Hi Tututu,
I am not sure which Flag M2 affinity gel you are using, but if you are using the one from Sigma, the gel can be easily recycled and reused. After elution with the Flag peptide, simply wash the gel couple of times in buffer then store in glycerol with azide. For a more detailed protocol, check out the Sigma website...and look for instructions on Anti-Flag M2 Affinity Gel Freezer-safe (product No A2220). Good luck on your experiment!
tututu 写到: Does someone know how to regenerate Flag M2 agarose?
After the flag M2 agarose bound with desired protein was eluted by Flag peptide, how to regenerate the Flag M2 agarose?
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- 帖子:459Hi Tututu,
Last time I did this was several years ago, so I can't recall the exact protocol right now. However, like I mentioned previously, just check the published protocol on the Sigma website and follow that. You are right, washing the gel with mere buffer will not remove the petide from the gel. You have to first do a standard acid elution with glycine to remove the peptide (the antibody interaction is pH-dependent). For sure, removal will NOT be efficient. That's I try to use fresh beads whenever possible. Good luck.
Tututu 写到: Did you do it by yourself about the regeneration of FLAG M2 agarose beads? i mean, you know, the antibody (M2) is attached covalently to beaded agarose. The peptides will be bound on the beads instead of the protein tagged by Flag. I am not sure that if simply washing the gel couple of times in buffer could remove the peptides on the beads. please give me the details of the protocol if it is easy for you. thank you very much.城市驾驶学校
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