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Normalise cell count for protein analysis


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#1 Chack

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Posted 14 August 2018 - 05:02 AM

Hi,

 

I have performed an ELISA to analyse protein concentration of a gene from multiple animals with different variations within the gene. However, I need to normalise the protein concentration to cell number, as, variable numbers of cells went in to the assay for each individual. Can anyone tell me the calculation for this. 

 

Thanks



#2 bob1

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Posted 14 August 2018 - 06:23 AM

I see you mention "animals" - are these from a solid organ or blood?

 

If it is blood, you can do counts quite easily using a hemocytometer. Solid organs are more difficult, unless it is something soft like the thymus or pancreas, which you can gently grind to release cells.

 

Couldn't you normalize to protein content of the lysate?



#3 Chack

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Posted 14 August 2018 - 10:56 PM

Hi,

 

Thank you for responding. I have counted the cells and I have a total number that I put in to the assay for each animal (primary macrophages), and I just wanted to uniform the number of cells to and adjust the protein concentration accordingly, so that the amount of protein is indicative of the function of the variation in the gene from half of the samples, instead of the amount of cells that went in. 



#4 bob1

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Posted 15 August 2018 - 05:38 AM

So you are looking for the calculation you need to normalize to cell number?

 

Ideally you would do this at the lysis level, where you would add a volume of lysis buffer dependent on the number of cells, perhaps 1000 cells per ul, or something similar.

 

However, you should be able to normalize to number as a form of concentration. I'm assuming here that you put a set volume of lysis buffer to a variable number of cells. 

 

Conc =Number of cells/Volume of lysis buffer (this would be for your initial sample, not when it goes onto the ELISA)

 

You would then normalize to a certain value (maybe the lowest concentration of cells/ml, or an untreated control) by dividing each sample by that value.






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