Hi everybody,
does anybody have reliable protocol for measuring the protease activity? We would like to establish the protease activity from laboratory compost experiment (made from sawdust) and currently used protocol is not working.
The input material is wet sawdust and we probably use buffer which is not working properly with this material (PBS buffer pH 8 with Triton X-100). Then azoalbumin is added, mixture incubated at 37 °C, the reaction is stopped by 10% trichloric acid, centrifugated, add 1M NaOH and absorbance measured 440 nm. The measured absorbance is from 0-0,004, there is a possibility there is a low level of proteases, but we would like to proove the occurence/absence in our sample with different protocol.
Thank you.
Susan
Hi Zuzana,
Please follow the link below that might give you some help regarding measurement of protease activity..
https://www.sigmaaldrich.com/technical-documents/protocols/biology/protease-activity-assay.html
Hi Susan :)
how about some positive control? How long is the incubation of the proteases with the azoalbumin? vWhat about changing incubation temperatures? Compost is usually rather hot, so maybe try to increase the temperature?
Good luck.
Hi Zuzana,
Please follow the link below that might give you some help regarding measurement of protease activity..
https://www.sigmaaldrich.com/technical-documents/protocols/biology/protease-activity-assay.html
Thank you both. :)
I inhereted this experiment after master student, so I am following the procedure for the next 4 weeks (already running for 6).
The temperature of compost is 58 °C and I guess I should have finish the experiment with the same temperature. The incubation with azoalbumin is 30 mins.
They were also measuring esterases and lipases activity (different protocol) and it was working well. So I would like to try another approach for the proteases and do both - new and the old protocol.
I'd try 30°C, 37°C and 50°C for 30 min, 2 hrs and overnight. That will be nine samples and will tell you, whether there is any change depending on temperature and incubation time and thus whether it is working or not.
Now that I'm looking at it again, you resuspend the sawdust in PBS and then what? Extract and centrifuge and add the azoalbumin? You should be adding only small fraction of the extract to the reaction mixture. Any idea what proteases to expect? Whether acidic or basic? Because for acidic proteases the pH 8.0 is obviously too high.
In addition to above, do you have some of the exact same batch of sawdust that the previous student was composting? You might try composting both that batch and your batch of sawdust in parallel, using the exact conditions used by your predecessor, and check for protease activity. You will find out if the lack of protease activity is due to a difference in the sawdust.
The azo-method is tried and tested, more modern variations use fluorescent substrates rather than azo-dyes for increased sensitivity. Also neat are zymograms (see DOI:10.2174/187220809789389162 for a review).
Are you interested in intracellular or secreted proteases? In the latter case, the neutral pH of PBS may not be appropriate, I'd measure the pH of the compost and use that for guidance.
The "ripeness" of the compost may also have bearing on the content of lytic enzymes.
Dear Zuzana
do you already known the specificity in terms of aa residues of your proteases?
There are commercially avaialble several kits for test proteases with unknown specificity eg:
https://www.thermofisher.com/it/en/home/life-science/cell-analysis/fluorescence-microplate-assays/microplate-assays-enzyme-activity.html
https://shop.jpt.com/2-Enzyme-Substrates/9-Enzyme-Substrate-Sets/101-Protease-Substrate-Set.html?_ga=2.102753857.114279421.1591705929-611895701.1591705929
in case you already know your protease specificity, you can buy quenched peptides suitable to follow peptide digestion using fluorescence monitoring, as happen in for the substate SET of JPT, or you can incubate the proteases with the recombinant protein substrate and monitor its degragarion over time by SDS-page, SEC or other tecqniques.
good luck
Manuele
Zuzana Šedrlová
Hi
I suggest you to have a look on the following protocol will ceratinly give you insight about the protease activity.
https://www.sigmaaldrich.com/technical-documents/protocols/biology/protease-activity-assay.html
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