I am running a Bradford protein assay and my protein values are falling beyond the standard curve. I am already using 1uL of sample in the standard microplate assay. Is there a way to extend the standard curve or dilute the sample to get the value within the desired range?
Hello Victoria
The protein concentration of an unknown sample is calculated from the standard curve that is generated form the known protein standards. If the protein concentration of the unknown samples lie beyond the linear range, you will have to dilute the samples. You can dilute each of your samples 10-fold i.e.1 part of the unknown sample and 9 parts of buffer used in the assay. Since you are using 1ul of sample which is too small a volume that could result in an error while pipetting, you can dilute your samples as follows :
10 ul of unknown sample and 90 ul of buffer. Perform the assay and determine the concentration of the unknown sample from the standard curve.
For example: If you obtain the value of x mg/ml for your unknown sample from the standard curve, you should multiply the obtained value by the dilution factor 10 since the unknown sample was diluted 10fold. So the concentration of the undiluted unknown sample would be 10x mg/ml.
If your unknown samples are highly concentrated, you can further dilute your samples 100 fold and follow the same steps as mentioned above.
All the best.
Bradfords response is not linear, hence do not do anything outside the curve. Dilute as recommended
The BCA assay is good as long as you're not working with denaturants. Pierce has a kit for it that decreases that effect, too. Getting under 100ug/mL is pretty tough for that assay, though. If you can compare the assays, we've found BCA gives a lower detection limit than Bradford. We use it routinely for ELISA quantification in our vaccine development work.
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