We wish to find the conc. of our antibody in a solution which is often low (2-6 ug/ml). UV Spectrometry does not work well, since we need to dilute it and need to know the extinction coefficient (so nanodrop also becomes problematic).
I was confused between bradford and bca assays, and was not able to decide, which one to go with. I will be using TSH monoclonal antibodies.
You could use the Bradford assay on such a dilute solution of antibody, but you will have to modify the procedure. Use the 5X concentrated reagent from Bio-Rad instead of the usual 1X reagent. Add 200 µl of the 5X reagent to 800 µl of dilute protein sample. Use purified IgG as the standard. Make sure the buffer is the same in all the samples, blank, and standards. If the samples contain anything that interferes significantly with the Bradford reagent (such as detergent), then this will not work.
Another approach is to concentrate your samples by TCA precipitation. The pellets can then be used for the BCA assay or the Lowry assay. To 1 ml of sample, add 100 µl of 0.15% sodium deoxycholate followed by 100 µl of 72% trichloroacetic acid. Let stand on ice for 10 minutes, then centrifuge at top speed of microcentrifuge for 10 minutes. Carefully and thoroughly remove the supernatant, and the use the pellet in the protein assay.
The extinction coefficient for IgG is 1.36, so if your antibody is pure, you can concentrate 10 fold, and measure absorbance at 280nm and divide the value by 1.36 to get the conc. in mg/ml. Thermo has a TechNote for using Nanodrop for IgG measurement:
http://www.nanodrop.com/Library/T010-NanoDrop%201000-&-NanoDrop%208000-Protein-Measurements.pdf
Are you sure that the antibody preparation does not contain any other proteins like serum albumin? Dilute protein solutions are subject to aggregation or binding to vessel wall, hence non-reactive proteins are often added to stabilise the antibody. In that case, of course, a simple protein assay would not help, you'd need to titrate the activity of antibody against a known standard.
I have been trying to read a bit about this (classes, thesis, projects, students, papers, etc, our daily battle...) and I have read that the A280 method rely on the amount of tryptophan and phenylalanine present in the protein. Been searching, and the papers that shows the amino acid characterization of antibodies shows very low F and W. This leads me to conclude that its not a very good way to measure antibody, using NANODROP. Am I mistaken here?
Andrei Moroz : Cystine also absorbs at 280 nm, although the extinction coefficient is some 10-fold lower than that of aromatic amino acids. It is possible to calculate the molar extinction coefficient of a protein from its amino acid sequence (doi:10.1111/j.1432-1033.1986.tb09653.x: epsilon_{280} = 5550*Trp + 1340*Tyr + 150*Cys [l/mol cm], doi:10.1016/0003-2697(89)90602-7}
epsilon_{280} = Tyr*1280 + Trp*5690 + Cys*120 [l/mol cm] for the denatured (6 M Guanidinium) protein.
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