Dear Isabella

Did your protein contains aromatic aminoacids (eg Tryptophans or tyrosines?)

UV quantification of a protein (with both nanodrop or standard cuvette spectrofotometer) is generally performed measuring the absorbance at 280nm due to the presence of those aminoacids.

Following the lambert beer law, the A=ebc and therefore the c=A/(e*b) where e is the molar exctinction coefficient that could be calculated at

https://web.expasy.org/protparam/ pasting the protein sequence.

The detection limit, depends a lot from the number of aromatic present in your sequence and the absolute value of the extinction coefficient.

For full lenght monoclonal antibodies for example using nanodrop, you can obtain good signals with concentration >0,1mg/ml.

The main interferences are due to all the molecules that are able to absorb at 280nm (eg oxidated DTT, ) but generally you can have an idea of it by determine the absorbance curve of the protein buffer alone.

In your case, i think that you can have, or a too low protein concentration or a protein with no aromatics that therefore is not detectable with this approach.

good luck

Manuele

Dear Manuele , thank you for your answer! It is a complex sample so normally should contain aromatic amino acids, but I am not sure which proteins are in there yet. I re-tried a Nanodrop experiment with more material, and this is what I got now. But again, I have this peak at the beginning of the graph, what does this mean?

Dear Isabella

i think that in your case the problem is the amount of protein that it is low and the absorbance at 220 is due to some other contaminants.

The peak at 220nm is probably due to the presence of some low mw peptides does not contains aromatics or some components of the buffer (which buffer are you using?)

To remove it, you can try to do the blank with the out of the protein concentrator (if you are concentrating your sample using ultrafiltration of similar)

Ciao

Manuele