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Calculations for ELISA of tissue lysate


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#1 smr86

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Posted 16 January 2014 - 02:52 PM

I'm new to performing ELISAs on tissue lysate so please forgive this basic maths question!!

 

I've performed a Bradford on my samples and now I'm at the maths stage of figuring out how much protein to assay and how to calculate the necessary dilution.

 

For example, I diluted my samples 1:100 for the Bradford, from the standard curve one of my samples was calculated at 29.41 ug/ml which becomes 2941ug/ml when I factor in the dilution, and then converting this to ug/ul I get 29.41ug/ul.  I obviously want to assay as much protein as possible ( I was thinking 100ug? per well)  So for 100ug protein, I need to use 33.93ul of my sample, but for the ELISA, I need to add 100ul sample per well, so do I just make up the remainder (66ul) with RIPA buffer or will this dilute my carefully calculated protein concentration? Or have I gone about my calculations in a completely wrong manner? 

 

Glad of any advice rolleyes.gif



#2 mdfenko

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Posted 17 January 2014 - 05:07 AM

you wrote it wrong (2.941ug/ul, not 29.41) but calculated correctly for 100ug.

 

you should make the volume to 100ul with buffer (i'm not sure if ripa would be the better buffer, in what buffer is the protein?). you want 100ug/well, final concentration will be 1ug/ul (100ul/well).

 

your calculated concentration is important only for knowing how much to add to each well.


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#3 smr86

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Posted 17 January 2014 - 06:23 AM

Thanks mdfenko.  Well spotted typo, its correct in my Excel file anyway.  The protein is in Krebs so yeah I guess I should make up the balance of the 100ul with Krebs then






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