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How many cells needed for co-IP and mass spec

co-IP mass spec IP

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#1 Elizabeth8

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Posted 09 December 2011 - 05:22 PM

How many cells do I need for a co-IP followed by mass spec to identify binding partners?

I have a stable cell line expressing a particular protein with a FLAG-HA tag. I would like to immunoprecipitate this protein (using Sigma's FLAG-HA tandem affinity purification kit). I would then like to use mass spec to identify all the binding partners of my FLAG-HA tagged protein. Approximately how many cells will I need to obtain sufficient protein for mass spec identification?

Thanks!

#2 bob1

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Posted 11 December 2011 - 12:24 PM

It depends on the expression of the protein, I would start with about 10^9 cells and work up from there.

#3 Elizabeth8

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Posted 15 December 2011 - 03:38 PM

So if one T75 flask has about 1x10^7 cells, then I would need 100 flasks to get 10^9 cells! Or about 50 flasks if they are the T160 size. Do people really use that many flasks?

#4 bob1

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Posted 17 December 2011 - 01:09 PM

If you want to identify all the binding partners, you will need a lot of protein to get detectable bands reproducibly.

#5 Ketil.Pedersen

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Posted 21 December 2011 - 02:33 AM

Cell numbers vs protein amounts
It varies between cell types but for SW480 cells 0.05 g is approx 10 million cells.

Co-IP for detection with WB or silver staining requires 0.05g to 1.5g of cell sample. For detection with coomassie or MS 1-15g of cell sample

Ketil Winther Pedersen
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