After running Elisa, i ran gel and western to confirm the results. I have a question with the densitometer. I have the problem to quantitate the concentration of the protein based on the bands in the SDS PAGE and WB : the volumes load into the lane. I expected the difference concentrations when I loaded with different volumes (20ul, 10ul, 5ul, ...). It was neither comparable with ELISA nor Forte. 50% lower with 20ul, 50% higher with 10uL.
Protein loaded on each band was 2 ug less. The max volume was 20ul.
Could you kindly give me some explanation ?
https://openwetware.org/wiki/Protein_Quantification_Using_ImageJ
https://www.youtube.com/watch?v=t9k8HFL88kk
Densitometrical analyses of Western blot pitures are rather not exact and not suited to quantify protein conentration. I would not anlyse Western bots via "semi-quantitative" methods like this. I would prefer and concentrate on quantification via ELISA.
Thank you so much, Bhairavi, Andreas. I had control at 3 different concentrations, then made a standard curve based on intensity. From that one I calculated the concentration of the unknown. I did not expect the results the same as the Elisa. Just want to see the correlation. Thank you
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