发表于: 7/22/2004 14:44 发表主题: Re: Does someone know how to regenerate Flag M2 agarose?
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Hi 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?

发表于: 7/22/2004 16:26 发表主题: Hmmmm...
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Hi Tututu,

Have we ever met? How do you know that I don't like a Biochem major wink In any case, I will take your comment as a compliment.

BTW, was the info useful at all?


tututu 写到:
what do you major in? biochemistry?
You don't look like that kind of student.

发表于: 7/22/2004 17:00 发表主题: ...
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Hi Tututu,

Seriously, have we ever met? Literature and art are hobbies, not my main profession smile

tututu 写到:
i get the information with your help, but i don't know if it works. you look like those student who major in literature, that kind of things.............

发表于: 7/23/2004 11:40 发表主题: Check the published protocol
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Hi 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.