Hello, I would like to get help! I want to measure the protease activity of pancrease powder. I prepared 10mg/ml stock solution from the pancrease powder and I used 100ul from that and I added 900ul 1% casein solution. The sample was incubated at 37°C, 30mins (in 50mM Phosphate buffer, pH8.0) and after the incubation 1ml 10% TCA was added. I left the sample at room temperature, 20mins after I centrifuged it (8000g, 5mins) and the protein concentration was measured from the supernatant by Bradford and Biuret. But I wasnt able to detect the protein concentration, because there wasnt enough color effect. I dont really understand why, because the pancreas stock solution is very concentrated. Do you have idea what can be the problem with my assay?
Thank you so much!
(I attached my protocol.)
To me the protocol looks fine. Is it that you are not getting color at all in any reaction or it is too low to detect? One thing first confirm is if the pH is affecting your protein estimation. If that is not the case, second thing I would check is on white background if trend in color is seen, this would tell you if enough enzyme is being used or not or if the enzyme still has activity or not, based on which you can play around the enzyme and substrate concentration. Another thing is make sure that your are adding excess of substrate. If your extract is too concentrated with enzymes, it would mean that your substrate is getting exhausted, so you may need to add several folds more substrate. If all these things are fine, one more thing I would do is I would estimate the protein at 280nm. This wold tell if the estimation method is suitable for assay conditions or not. Last thing, for preparing blank, I would add enzyme and substrate to TCA solution to ensure reaction inhibition in blank. All the best.
Erna Szabo I feel you just misunderstand. BCA method is used to measure peptide concentration, why you used Biuret method? The sensitivity is different 2 orders. I recommend finding (1)the merit and demerit of assay method, (2)sensitivity of assay method, (3)what interferes the assay. i.e. pH, components and so on.
I tried the all blank because of the background. My sample is a mix. :) I used more and more enzyme concentration and another substrate concentration. I got good result, measuring the 280.
Michitoshi Watanabe, we don't have BCA kit or reagent to measure the content by it. But the BCA is used for quantitation of total protein in a sample. The principle of this method is that proteins can reduce Cu+2 to Cu+1 in an alkaline solution (the biuret reaction) and result in a purple color formation by bicinchoninic acid. So, it was the cause why I tried to use the Biuret.
But A280nm seems to be the best.
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