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Scaling up reagents for large scale immunoprecipitation

protein A/G antibody immunoprecipitation mass spectrometry

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#1 cancerous

cancerous

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Posted 30 December 2012 - 03:06 PM

Hi all,

I am planning to do large scale IP for mass spectrometry sample prep.

I did preliminary IP with one 10cm plate of cells using 50ul protein A/G (sc-2003, Santa Cruz) and 2ug antibody to pull down. I did see about >90% protein of my interest is pulled down, and need to now scale up for mass spectrometry.

I have 200 times more cells (200X 10cm plates), so wonder how much antibody and protein A/G I need to use. I think it is not necessary to use 200X every reagent. Does anyone have any suggestion? Does anyone know how much volume of protein A/G can bind to how much ug antibody?

Thanks in advance.

Cancerous

#2 Curtis

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Posted 02 January 2013 - 05:16 AM

I only used 20 ul of protein A/G. For my protein G I used only 1 ug of antibody and rotated overnight to bind. I think you need to consider the cost rather than the amount you can actually add. last time I collected cells from 3 x 150 ml flasks but I only used 30 ul of protein G and same amount of antibody.





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