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How to detach an antibody from cell surface


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#1 mrvica

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Posted 26 January 2010 - 03:56 AM

Hi!
As I have allready asked in topic title, can you tell me a good way to detach antibodies (after treating my cells with them for a period of time) from the cell membrane. The reason for this is next: i need to see if the treatment has caused the change in antibody production by the cell itself, and in ICH, I need to detect only cell-derived antibodies and not the treatment antibodies, if you know what I mean (since the tretment and produced antibodies are human origine, my secondary antibody recognizes both of them, and that`s the main problem). I have tried with extensive washing with PBS, but that doesn`t help a lot. Do you think that the pronase or trypsin treatment woud help?

Thanks!!!!

#2 sgt4boston

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Posted 26 January 2010 - 07:47 AM

surfactant or pH change?

#3 mrvica

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Posted 26 January 2010 - 11:46 PM

First of all, thank you for answering me :) . I was thinking to try with 150mM NaCl in bidestilated water, pH 2.5, hope that works. But if it doesn`t, what surfactant did you have in mind?
Puno pozdrava!!!
(that is: Best regards, in my language :D )

#4 sgt4boston

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Posted 29 January 2010 - 04:39 AM

Can you try all 3 treatments in parallel; low pH, enzyme, surfactant (tween 20/sds/triton x100)?

#5 bachai

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Posted 31 January 2010 - 02:32 PM

Not sure how would you preserve your cells at harsh conditions required for dissociation of Ab from the cell-bound Ag.
Can you try reduce the treatment Ab to F(ab)2, then use anti-Fc conjugate to detect produced Ab?




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