ELISA standard curve - approximate protein concs to use? - (Oct/26/2007 )

Hi all,

I'm just about to run an ELISA. I have done a titre of my primary and secondary antibody to determine the concs i should use, primay will be 50ng per well.

I now need to produce a standard curve for my protein. It has been purified, and the monoclonal is VERY specific for it. we have decided to do a competitive ELISA.

What i would like to know, is what is the approximate range of protein concentrations i should use to construct my curve? I really have no idea... 1mg and lower? 1ug and lower?

Thanks for any help...

L

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Hi Flour,
even i have done a competitive curve by ELISA recently
as you mentioned that your antibody is specific i suggest you to start the curve from 1ng to 500ng to give a complete curve
all the best

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hi,
i wud agree with leelaram, but i wud do one more exp, in which use different concentrations 10,100,1000,10000ng. based on the results u can choose the range which is suitable to produce a curve.

gud luk
sravan.

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I agree with leelaram and sravam. When we perform an ELISA we done the next steps
1) Several concentrations of antigen and a constant concentration of primary and secondary antibody ---> look for optimal antigen coating concentration
2) Several concentrations of secondary antibody and a constant concentration of antigen (optimal) and primary antibody ---> look for optimal secondary antibody concentration
3) Several concentrations of primary antibody and a constant concentration of antigen and secondary antibody ---> look for antibody titer.

When we change the source of antigen or secondary antibody, or polystirene plates we perform again these ELISA to have the best conditions to perform ELISA

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