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hey guys,what do you think the Anti-Flag M2 agarose beads is made of?


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#1 AllenChiu

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Posted 16 March 2010 - 09:11 PM

hey guys,what do you think the Anti-Flag M2 agarose beads is made of?
If it is made of regular agarose,why wouldn't it melt when I boiled the beads with loading buffer?
Well,actually I want to make some homemade agarose beads,which haven't been crosslinked to any protein A/G or antibody.
Because I suspected the non-specific binding of IP is caused mainly by the agarose part of the beads,instead of the protein A/G or antibody.
So I want to preclear the lysis with large amount of cheap homemade agarose beads?
Do you think it make any sense?What do you think?
Thanks

#2 NemaToStella

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Posted 16 March 2010 - 10:48 PM

Hey there, to be honest spending your valuable time on trying to make homemade beads will ultimately be more expensive for your PI than just using some cheap protein A/G agarose.
Alternatively, instead of using the anti-FLAG M2 agarose beads, why don't you try protein A/G Dynabeads in combination with FLAG M2 antibody? They have a much lower level of background binding.

#3 AllenChiu

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Posted 16 March 2010 - 11:18 PM

Hey there, to be honest spending your valuable time on trying to make homemade beads will ultimately be more expensive for your PI than just using some cheap protein A/G agarose.
Alternatively, instead of using the anti-FLAG M2 agarose beads, why don't you try protein A/G Dynabeads in combination with FLAG M2 antibody? They have a much lower level of background binding.

Well,I do have used Dynabeads.And it really has a lower level of background in Co-IP,I haven't use it for IP.
I suspected it because Dynabeads were hydrophilic?what do you think?
Well,have you use Dynabeads for IP-MS,how much beads per sample then?

#4 NemaToStella

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Posted 17 March 2010 - 10:03 AM

No, I haven't. I've used avidin agarose before which had a really bad background even after extended preclearing with generous amounts of avidin agarose beads before the actual IP. Switching to Streptavidin Dynabeads greatly improved this issue and the samples could be used for MS. Have to look up how much beads I used, can't remember right now.




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