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whole antiserum for indirect immunofluorescence?


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#1 BottegaVeneta

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Posted 13 December 2009 - 06:59 AM

hello,

i have this rabbit whole antiserum from sigma for my protein of interest. western blots are very clean, but i'm worried that when i do indirect immunofluorescence, i'm getting non-specific binding. for a negative control, can i use pre-immune whole antiserum from any rabbit, or does it have to be from the same rabbit that produced it in the first place? alternatively, can i pre-block the antiserum with (semi)purified protein expressed in bacteria as a negative control?

thank you!

#2 sgt4boston

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Posted 14 December 2009 - 10:12 AM

neg control can be any rabbit but should be same type of rabbit.

By pre-blocking do you mean adsorb out the non-specific abs? I think it would be best to purify your antibody of interest...best would be affinity purify.

#3 BottegaVeneta

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Posted 14 December 2009 - 10:19 AM

neg control can be any rabbit but should be same type of rabbit.

By pre-blocking do you mean adsorb out the non-specific abs? I think it would be best to purify your antibody of interest...best would be affinity purify.


Yes that's what I meant by pre-blocking. I don't have enough for affinity purification because they only give 200 uL. I think my best option right now is to use whole antiserum from rabbit, but I wasn't sure if it HAD to be preimmune serum from the same rabbit that produced the antiserum. Thanks for your help!




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