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How many B cells do I need to get a 1mg of protien


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#1 SaraK

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Posted 06 November 2009 - 09:45 AM

Hi all,
I would like to purify mouse B cells and look at protein expression by IP western. I'm trying to back calculate how many mice of each strain I will need for my experiment. Generally I like to have a couple of mg of protein to work with when I setup my IP's but I don't know how much protein to expect per B cell. Does anyone have any experience making protein lysates from purified B cells?

Thanks
SaraK

#2 Dr Teeth

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Posted 06 November 2009 - 11:41 AM

Hi all,
I would like to purify mouse B cells and look at protein expression by IP western. I'm trying to back calculate how many mice of each strain I will need for my experiment. Generally I like to have a couple of mg of protein to work with when I setup my IP's but I don't know how much protein to expect per B cell. Does anyone have any experience making protein lysates from purified B cells?

Thanks
SaraK


I don't know much about B-cells, but many mammalian cell culture lines average around 300 pg/cell of total protein, but, this of course varies by cell type/size, with smaller cell types averaging only ~150 pg/cell. I would make a conservative judgment that you would need at least 3x10^6 and as much as 1x10^7 cells for 1 mg of protein.

However, I also found a publication suggesting that B-cells have around 6.4 ug/10^6 cells, so you may want to go by these.

see Table 1 in:
B cell proliferation following CD40 stimulation results in the expression and activation of Src protein tyrosine kinase
Sonia Néron1,2, Garnet Suck3, Xue-Zhong Ma3, Darinka Sakac3, Annie Roy1, Yulia Katsman3, Nathalie Dussault1, Claudia Racine1 and Donald R. Branch3,4
International Immunology 2006 18(2):375-387; doi:10.1093/intimm/dxh377
http://intimm.oxford...l/18/2/375#TBL1

Science is simply common sense at its best that is rigidly accurate in observation and merciless to fallacy in logic.
Thomas Henry Huxley

#3 miBunny

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Posted 06 November 2009 - 06:06 PM

Personally, I'd run a quick test on a junk mouse. Greater than 90% of the splenic cells will be B cells or T cells. B and T cells are almost the same size and will have roughly the same amount of protein (cytoplasm to nuclear ratio is pretty similiar). Lyse different amounts of total cells and calculate the amount of protein. That will get you within the ball park for protein quant vs cell number.

As for how many cells you will get, that will depend on what you are purifying... B220+ or mature follicular B cells, ect (there are lots of different B cell populations) In general, roughly half the splenic cells will be B cells of some sort (marginal zone, T1, T2, or Folicular)

Also, the amount of protein you get will partially depend on how you are lysing your cells. So it is best to test it out in your own hands.




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