Dear colleagues,
I would like to analyze samples of human renal cell carcinoma for HIF1A by using sandwich ELISA kit (LS-F4224). However, I am not sure how to dilute the samples. The range of standard curve is 0.157 ng/ml to 10 ng/ml and the average protein concentration per sample is 4 400 microg/ml. Can anyone help please?
Hello Daniela Vargová
It depends upon the concentration of the target protein in the sample. I would suggest you try a few dilutions initially like 1:10 and 1:100. This will give you an idea about the level of target protein in the sample. If the absorbance value of the sample is beyond the standard curve you will have to dilute the sample further. Choose that dilution which lies within the range of the standard curve preferably, in the linear range of the curve. The linear range of the standard curve gives the accurate concentration of the analyte. You then multiple the concentration obtained by the dilution factor.
Best.
Thank you very much for your answer Malcolm. But what if I do not know the concentration of the target protein? And is it not too much to dilute the sample 1:100? Because I assume there is not much HIF1A in healthy kidney tissue and I am afraid it would not measure the target protein when using dilution 1:100....
So you try the sample as neat that is without dilution, if you are expecting a low concentration of the analyte.
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