I am going to try to determine the activity of protease in soil samples using a azocasein as a substrate. I found references in literature to the works of Macura and Vagnerov (1969) and Charney and Tomarelli (1947), but I have a few questions. I would be grateful for the some advice.
1.) At what temperature should the samples be incubated after the addition of an azocasein solution? I found a version with 30*C and 37*C...
2.) Spectrophotometric measurements should be made at 430 nm or 440 nm?
3.) Is a soil sample with all reagents excluding the substrate used for determination as blank spectrophotometer? In this case, if I have 7 different soil samples, do I have to prepare 7 control for them?
Dear Karolina,
Ad 1.) You should carry out an experiment at both temperatures, or better at wider range of temperatures, it is hard to predict where is the optimum. This may also vary in your soil samples, so finding temperature optima in all 7 samples may provide interesting results.
Ad 2.) This should be specified by the supplier of azocasein (e.g. Sigma recommends 440 nm)
Ad 3.) Determination of blank parameters requires more insight into the experiment setup, it is probably good idea to consult this with your supervisor.
Hi,
1.) The incubation temperature depends on the temperature optimum of your proteases. Since your sample is soil i would test different temperatures. For a first check i would suggest 20 °C, 30 °C and 40 °C.
2.) I normally measure at 440 nm.
3.) I recommend at least a positiv control with a protease solution (eg. Trypsin) and a negativ control, where you do not expect any protease activity.
I hope i could help.
Best wishes,
Philipp
Dear Jan,
thank you for your answer.
I am preparing my own experience, so I use information from literature. I was looking for procedures for the determination of proteases in soil to create a universal procedure for various soil samples. In the future, I am going to analyse more than 7 samples, so I'd like the control to be easy to prepare for different experience.
Dear Philipp,
Thank you very much for your answer.
Thank you for your practical advice! I will take advice on wavelength and control. And I will try to incubate at least two different temperatures.
1. The temperature range of 25 and 40 degrees Celsius is sufficient for incubation.
2. I recommend you use the wavelength of 440 nm.
3. One single positive control can be used for the assay.
A negative control could contain the protease added after addition of the 10% TCA (termination) to the buffered azocasein. That way the negative control & test will contain the same components but proteolytic activity will be absent.
- Badges
- Science topic
- Similar topics
- Social Science
- Media
- Journalism