Dear Kana Ng

calculation of protein concentration may often change on the basis of the methods and unfortunatelly there is not universal best method but which is the most reilable method it depends from protein properties, concentration, buffer composition and protein that you are using to perform calibration.

For example Bradford or BCA using the BSA as standard underestimate the concentration of monoclonal antibodies and A280nm it more reilable, but of course if you are replacing the BSA wirh a mab with known concentration to for the calibration, also BCA or Bradford estimation becomes reilable.

In your specific case, it is strange that you obtain an higher value with ABS280 since your protein seems to do not contain any Tryptophan and it extiction coefficient is therefore quite low and the meausmente of A280 could be tricky.

calculation with https://web.expasy.org/protparam/

Extinction coefficients: This protein does not contain any Trp residues. Experience shows that this could result in more than 10% error in the computed extinction coefficient. Extinction coefficients are in units of M-1 cm-1, at 280 nm measured in water. Ext. coefficient 7575 Abs 0.1% (=1 g/l) 0.166, assuming all pairs of Cys residues form cystines Ext. coefficient 7450 Abs 0.1% (=1 g/l) 0.163, assuming all Cys residues are reduced

Therefore i suppose that you high valuse could be due to some contaminants, for example:

Which is the protein buffer?

Which is the ration 280/260nm of your sample?

best

Manuele

Dear Kang,

Can you mention the lysis buffer you use? And which firm?

There must be an interference in your solution which occurs at 280 nm but not in the BCA assay. It is better to confirm this by blank subtraction (analyzing reagent blank). At this point, it may be better to share your purification strategy and the buffers you used...

Thank you so much for your responses Manuele, Mohamed and Ismail!

The lysis buffer that I used contains Na2HPO4, NaCl, TritonX-100 and beta-Mercaptoethanol. My elution buffer has Tris-HCl and Glutathione since I used Glutathione Sepharose resin to purify the protein. I then dialyzed the eluted protein against 1X PBS for 3 times. The volume of 1X PBS used each time is 50 times the protein volume. The blank i used for measuring the background at 280nm is 1X PBS.

So you think its likely that there're some small molecules (beta-mercaptoethanol in this case?) that interfere with the A280 signals or in other words, 3 times dialysis might not be good enough? is there a chance that the BCA assay is not that sensitive when it comes to detecting Tau441 since Tau441 sequence has no Tryptophan, only 1 Cysteine and a couple of Tyrosine?

Kana Ng

BME interferes with BCA and some other protein quantification methods. On the other hand Triton-X is a detergent interferes with measuring by 280.

In the attachment a list of compatibility with BCA assay.

For more information about the choosing of lysis buffer pls refer to the following article.

DOI: 10.1080/14789450.2017.1388167