diluting standards BCA assay - (Sep/19/2012 )

Hello,

I recently did a protein estimation by BCA assay which had a standard protein concentration starting from 25ug/ml - 2000ug/ml.

The spectrophotometer that I used for the assay does not give OD's above 1.0 and hence of the 6 diluted standards I prepared I was able to use only 3 of them from 25ug/ml - 100ug/ml after which the OD's was above 1.0 and the reader was not able to give any values.

My question is : Is it valid to dilute the standards that you have made from 200ug/ml - 2000ug/ml by 1:1 and than take the OD values and multiply it by the dilution factor (0.5 in this case). And than use the ODs obtained here and before (25ug/ml - 100ug/ml) and construct the graph to determine the concentration of the unknown protein sample?


If this is a valid assay, is there any reference that you can suggest for this, and if you think it is wrong to do this please provide an explanation for the same.


regards,
thekid

In general no, it isnt valid to dilute your standards and multiply back up.
What you can do is dilute your samples so they fall into the range of your valid standards, then multiply back up to account for the dilution factor.
(as in, you do a 1:10 of your sample so it falls in the range of the standards, then multiply the value by ten to get the actual concentration.)

You can repeat the assay with more dilute standards (it is unclear if you were intending to dilute the standards prior to rerunning the assay or post-assay which wouldn’t be ok). Having only 3 data points to base the standard curve on is risky particularly at the low end of the assay range so you need to have more low-end dilutions or an increased number of replicates.
But the first thing I’d do is have a look at the specro settings; chances are someone has set the limited to 1 for reasons that are not related to your assay. Reaching an OD of 1 with 100ug/mL does seem a bit high for BCA but that is not important as long as your samples are treated the same way as the standards.