I have just cloned and got a soluble expression(50%) of a thermophilic serine protease and did the heat treatment for purification at 70 and 80 degree with over 90% purity, and also have a strong activity with casein substrate during assay. But I wanted to confirm the presence of the recombinant protease which produced single band after 80 degree heat purification for Ihr and 15 mins . I want to perform proteolytic activity staining for the confirmation. I would like to know how to go about the proteolytic activity staining and protocols that will help me to achieve this . My recombinant protease is a highly thermostable serine protease with MW of 36kDa. Thanks
NB. The gel for the heat treatment purification is attached, the 3rd line from the ladder is the 80 degree heat treatment
Suleiman Allison Hi, you can incorporate casein into the gel and do a zymography. An active proteinase will show a clear band against a blue background where it comes to rest in the PAGE after protein staining.
You can either purchase these gels:
https://www.biocompare.com/24056-Zymogram-Gels/98744-Novex-12-Zymogram-Casein-Gel/
Or make them yourself by making the acrylamide solution 1% in casein.
You can prepare an agar gel that containing the desired concentration of casein on glass plate. And after the polyacrylamide electrophoresis run has been completed, incubate the polyacrylamide gel that contain the separated pro-tease band with agar that containing the casein ( i.e. sandwich them together face to face and rub them so they will not move & stay in their position).Then put this sandwich in the incubator for a period of time at 37C, if the separated band is protease you will notice that there is a clear zone on the agar gel in the location that facing your band as a result of hydrolysis of casein by the protease while the rest of the agar gel appear cloudy since it contain unhydrolyzed casein.
Good luck.
Hi Suleiman,
Of course you can detect activity of you enzyme using zymography.
1. Copolymeraze 0.03- 1% casein (try different amount) with polyacrylamide during gel casting
2. Prepare sample: mix protein sample with loading buffer without beta-mercaptoethanol (SDS can or cannot affect - also should be tested), incubate on ice 30 min. Use prestained standards!
3. Run SDS-PAGE at 4C - all buffers must be cooled, also put your electrophoretic chamber on ice (even better to use magnetic stirrer) or run in cold room
4. After running cut protein standards line thoroughly wash your gel with 2.5% Triton X100 for removing SDS (10-15 min, twice), wash with your buffer in which your protein is active
5. Add your buffer and incubate overnight (or you can also optimize the time of incubation) at desired temperature
6. Stain your gel with Coomassie R250, after destaining put together standard line piece with the rest of gel and make image. Of course MW will not be so correct (since you did not denaturate your samples) but anyway will give some information about possible molecular weight.
Good luck!
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