I ran a zymogram of protein samples but couldn't get the result. The gel was half stained and half destained showing no zone of hydrolysis. Why is it so? The protein zymogram was run but the gel was half stained and half destained showing no zone of hydrolysis. Why is it so?
It is most likely that the gelatine substrate has been degraded. When you dissolve gelatin into solution by heating it, make sure you do not bring it to boil. If you build, gelatine will be degraded into smaller peptides, and upon running gel, they will co-electrophoresed with your sample, resulting half of the gel with no-staining. To dissolve gelatin, heat it upto 80 degree C which is high enough to dissolve it and low enough to prevent it to degrade. Good luck!
Hello Sobia Ejaz,
The Zymography is only meant for Hydrolytic Enzymes, And for protease zymogram you should not add any reducing agent along with the loading Dye(S'd not boil the sample). After running time,incubate both substrate gel and sample gel in a buffered condition.
Dear Sobia Ejaz,
Generally zymogram is carried out after PAGE analysis to identify protein bands which shows the enzymatic activity. I feel your question is incomplete. Please mention for what enzyme you want to do zymogram analysis...?
It is most likely that the gelatine substrate has been degraded. When you dissolve gelatin into solution by heating it, make sure you do not bring it to boil. If you build, gelatine will be degraded into smaller peptides, and upon running gel, they will co-electrophoresed with your sample, resulting half of the gel with no-staining. To dissolve gelatin, heat it upto 80 degree C which is high enough to dissolve it and low enough to prevent it to degrade. Good luck!
In case of half stained and half destined, the possibility is that the substrate you have added might have settled to the bottom part. So check whether the solution is mixed properly and the solution should forme gel in quick time.
What protein are you working with? As Naresh says if you are working with a hydrolytic enzyme you may just have to optimise. For example if you have a soluble substrate this can be added to the Native gel, or to an agar plate on to which the gel can be placed to observe activity after incubation at the optimum temperature. Also you may need to modify a substrate buffer in which you leave the gel at an optimum temperature with agitation for a period of time. If you are running the zymogram from a denaturing gel (SDS-PAGE) you will have to renature the enzyme using a detergent, such as Triton X-100. Hope this helps.
Hello,
Be sure to rinse the gel thougroughly after running and before incubating with the developing buffer to completely remove the SDS. Otherwise you won't see the proteolytic activity. I used 2.5% Triton X-100 in 50 mM Tris HCl pH 7.5, followed by distilled water (for gelatinases).
Good luck!
Please Could you clarify your question?
When you prepare the gel acrilamide, the added sustrate must be an homegenous mix. All the surface of gels must contan sustrate. Your samples must be prepared in non reducing conditions, you need native protein (proteases). You only detect the sustrate degration and the band coorespond with the electrophoretic migration of the enzyme in study. If you like you can review this paper ARTHRITIS & RHEUMATISM
Vol. 43, No. 12, December 2000, pp 2807–2817, my Email is included in the paper. All the best
I agree with Yoshifumi Itoh, you probably warmed up the gelatin, you should dissolve it at 37-40 ° C
I completely agree with the answer by Yoshifumi Itoht and by Naresh Manickam. In addition, based on my experience I suggest to use fresh-prepared gelatin.
Hi,I also got the same result as yours. Along with above answers, I found the temperature raising during the SDS-PAGE run, also leads to this type of half stained and half unstained gels.
Hi Sobia Ejaz, it might also depend on the kind of substrate that you are using. For instance, if you use casein (not pure beta-casein), it can happen that you get a half not stained (or less stained) and a half stained due to the migration of the other forms of casein. This issue can be solved just by doing a pre-run of 20-30 minutes (or less) in order to get an uniform background. Also, the degradation of the substrate might influence the result as well.
Hello! for zymograms, I dissolve 0.2% gelatin at 37 ° C with vortexing until observing the disappearance of lumps at this temperature no proteolysis of the gelatin is dissolved homogeneously. Greetings!
Thanks to all of u. Basically the protocol I followed didn't give the details of developing buffer. Moreover, it didn't mention the procedure to dissolve the gelatin and to remove SDS from the gel using Triton X-100. Kindly any one give me a complete procedure of zymography in order to check protease activity.
I use this protocol:
Samples are mixed 3:1 with sample buffer (containing 10% SDS). Samples will be run under non-reducing conditions without heat denaturation on 10% polyacrylamide gel (SDS-PAGE) co-polymerized with 1 mg/mL of type I gelatin (stock solution 3 mg/ml in water). The gels will be run at 4°C and, after the SDS-PAGE, will be washed twice in 2.5% Triton X-100 - HCl 50 mM for 30 min each at RT and incubated overnight in a substrate buffer at 37°C (Tris-HCl 50 mM, CaCl2 5 mM, NaN3 0.02%, pH 7.5).
Try here
http://www3.szote.u-szeged.hu/hurodocs/downloads/biochemistry/HURO_zymo_practical_Kupai_final.pdf
Don,t fiorget to measure level of protein sample before running electroforesis, to make sure you will running sample contain enough protein or nothing. Good luck.
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