I have tried the Bradford protein assays for the estimation of unknown concentration using the BSA protein. I got a linear absorbance values using the BSA. However, when I applied this Bradford method to papain protein, it did not work at all. The color and the absorbance barely change at all concentrations even when the Bradford dye reagent was added. When I tried with lysozyme, it worked and different colors and absorbance changes were observed at different concentrations. So, is papain not a suitable protein to be applied in the Bradford method?
Dear Nay San Lin,
As you know that the Bradford protein assay is very sensitive and must be conducted with all care. If you do not know the approx. total protein conc. in your sample, run a assay with various dilution factor first in order to know the which dilution is working with Bradford protein assay. Plethora of studies have estimated papain protein using Bradford Assay without any intermediate interference after adding Bradford solution. For example, Identification and extraction of papain enzyme from papaya leaf in adigrat towen, northern Ethiopia. Journal of Medicinal Plants Studies 2018; 6(3): 127-130.
I would suggest you to crosscheck your sample and protocol once again in order to get the desired results.
Hope this may help you.
Best
Shail K. Chaube
Proteins differ in the sensitivity of their detection by Bradford reagent, but it is unlikely that papain is undetectable. Consider the possibility that your supply of papain is the problem. Does it contain a large amount of something that interferes with the assay? Is it actually a mixture of a small amount of papain and a large amount of something else?
Compare the papain and lysozyme sequences to get a roughly estimated response for each protein. If your papain sample is not very diluted it should be visible in Bradford assay but its signal may be less than lysozyme if its AA sequence has a low number of phe, arg, pro, try since coomassie binds to these AAs to produce absorbance in acidic conditions.
Professor Shail K Chaube,
Thank you for your recommendation.
I will read the paper you mentioned.
Hello Dr. Adam B Shapiro,
I just used a commercially available papain and mixed it with buffer. But, any differences cannot be seen even though the different concentrations are used. The same method was applied to lysozyme and it worked.
Dr. İsmail Emir Akyildiz,
Thank you for pointing out the AA sequences of papain and lysozyme. I will increase the papain concentration and try again.
My suggestion is evaluate absorbance (280 nm) of your sample (papain) and compare to albumin standard. Maybe you are working with low concentration.
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