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radiolabeling and co-IP with invitrogen dynabeads


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#1 research_xyz

research_xyz

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Posted 24 October 2009 - 01:32 PM

Hi everyone,

I am currently working with a radioactive labelled antigen and antibody (with His-tag) against it. I try to do an co-IP with my antigen + antibody and invitrogen Talon (cobalt) dynabeads. I did a control without the antibody and then my radioactive antigen doesn't bind to my beads. So they stay radioactive free. I tried elution with a 500 mM imidazole buffer pH 8 but the elution isn't 100 %. So my beads stay still radioactive bound. Other elution imethod with a low pH 4,5 acetate buffer wasn't better. I want to reuse my beads so I tried to remove the my antibody (and so the radioactivity) with :

- Stripping the beads once with 200 mM EDTA pH 7 and regenerating the beads with Co.
- Stripping the beads two, three and four times before regenerating them.
- Boiling in 2 % SDS, then stripping and regenerating them.

But my antibody is still bound to it with the radioactive antigen.

Does anyone off you have experience with other methods how I can remove my antibody from the beads and so making them radioactive free again.

Perhaps try with a NaOH solution ?

Thanks in advance

A desperate researcher




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