I ran gelatin zymography of my plant protein sample but there was no zone of hydrolysis. Moreover, the bands appeared similar as that of sds page bands. I ran sds as well as native zymography and both showed the same result. Is there is no protein activity in my crude sample?
Thank u..........
Forgive me if this may be obvious, but just for the sake of understanding I will ask you some obvious questions:
Did you prepare your sample in a buffer WITHOUT any reducing agent and APPLYING NO heating? Is gelatin the more suitable substrate for the plant proteases? Did you wash away any remaining SDS from your gel and incubate it in a suitable buffer at a suitable "pH+temperatire+time" combination for your proteases to work? Did you try to make a gel with a positive control (a purified ACTIVE enzyme)?
Also, I would check for total protease activity in your sample with a non specific substrate (such as azocasein), just to make sure that no degradation of the proteases occurred during protein extraction or storage.
As I said, that would help ME to understand, thank you for your answer.
Forgive me if this may be obvious, but just for the sake of understanding I will ask you some obvious questions:
Did you prepare your sample in a buffer WITHOUT any reducing agent and APPLYING NO heating? Is gelatin the more suitable substrate for the plant proteases? Did you wash away any remaining SDS from your gel and incubate it in a suitable buffer at a suitable "pH+temperatire+time" combination for your proteases to work? Did you try to make a gel with a positive control (a purified ACTIVE enzyme)?
Also, I would check for total protease activity in your sample with a non specific substrate (such as azocasein), just to make sure that no degradation of the proteases occurred during protein extraction or storage.
As I said, that would help ME to understand, thank you for your answer.
Adding to the 3 rd step from Mr. Vivek's answer. wash more than 3 times. Almost 10 to remove triton x100 and sds.
And I suggest some points
1. Conduct caseinolytic zymography also.
2. Do test tube assays for proteolytic activity. Where you can check for pH specificity and temp specificity.
These will help you.
And if you dont get then no harm in reporting that you found no activity.
Please check if using a suitable enzyme incubation buffer, especially with the cofactors, and not contaminated with EDTA / EGTA such inhibitors.
Hope it will help.
Thank You all for ur answer. I first added 0.5% gelatin in the resolving gel and polymerised. Then after running the sample (having no Beta-mercaptoethanol and not heated) soaked the gel in Triton X-100 for 40 min and then incubated the gel in incubation buffer (containing NaCl,CaCl2, Tris base and NaN3 having pH 8) for 20 hrs. Then stained and destained the gel. But still the gel showed no activity.
Oh then there is no activity is what you gotta report.
Check for caseinolytic activity as mentioned above.
If you want to see bands in 0.5% gelatin you need to wait for more time. According to our sample activity and we need to optimize the incubation time(You better go for 0.2 or 0.1 percent gelatin and incubate 12 hrs to 48 hrs, 0.5%--72 hrs etc.. at 37 degrees). In addition to that increase the Triton X-100 incubation time to 1 hr (30 min-wash 2 times-again 30 min in fresh Triton X-100) then wash 3 times with MQ to remove remaining SDS. Finally incubation time depends on your sample nature as well as your band intensity needed to you.
What is the pH of your plant extract?
Maybe you can try to perform the incubation at a pH that is close to the one your proteases are supposed to work at, in the cell?
By the way, could you please tell you what kind of matrix are you working with, and how did you extract proteins?
Thank you
This time I ran zymography with crude extract to check the protease activity. But still no results were found.I think I should follow Chandra's suggestion.
Thanku............
Getting no protease activity may be attributed to the following reasons:-
i. protease might have lost the activity due to denaturation or any harsh conditions that you might have used during your experiments.
ii. there might be inhibitors of protease present in the sample.
iii. optimum conditions for the protease to show activity might have changed in your case such as pH, temperature, ionic strength, substrate concentration etc.
Cross check the above basic factors, You may get your result.
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