Hello everyone, I worked with zymography during my PhD, in 2012. I prepared fish bile by sonicating the liquid sample for 5 min, removing lipids (using cleanascite) and removing salts (using vivaspin columns).
I used mini-gels and loaded 10 ug protein in each lane (didn´t use DTT or any other reducing agent, did not boil samples, did not boil gelatin). I used 0.1 % gelatin in the gels, and all solutions were prepared fresh. I incubated the samples for 13 hours and wanted to see ALL enzymatic activity of the samples, since it was my aim to characterize what was present in the sample.
The results were beautiful and published.
Tried the same thing with mussel Perna perna digestive gland and muscle tissue (same sample preparation protocol, sonicating, delipidizing and desalting, loaded 60-150 ug protein since gel is large). NO enzymatic activity whatsoever. Only difference was using large 13 cm gels. All solutions were prepared freshly, the gel was stained blue, but no digested area appeared (did not use positive control yet).
Any ideas?
- Too short incubation time?
- Too much protein (at 80, 120 and 150 ug the bands stained with coomassie, I assume this is too much protein, but 30-40 gave me no response so I tried to increase protein loads)?
- Too little gelatin for larger gels (still at 0,1%)?
- Sample preparation not OK? I DO NOT want to use protease inhibitors since I want to see ALL enzymatic activity in the samples.
Definitely repeat with positive gelatinase. Also, be sure to have cofactors (eg., Mg) present
it is a new gelatin or the same you use in 2012? maybe you could change that.
Do you use Brij 35?
Try to change the gelatin for caseine.
Use 10% Ready Gel® Zymogram Gel, 10 well, 50 µl #1611167 from Bio-Rad ($ 16.00) and loading 20 ug protein.
Good luck.
Have some news: Protocol is ok, did some fresh fish liver samples and saw hydrolitic activity. Used gelatin (stock, powder form) from 2012, but gel is stained, so seems ok... I use Triton X-100.
The samples of interest (mussel Perna perna), however, showed VERY LITTLE activity.. Some bands were slightly stained, do you think the problem is too much protein loaded on the gel? Or too short incubation time (about 18 hs)? Or perhaps how I prepared the samples (sonicated, delipidiez and dessalted them). Should I simply homogeneize them in a buffer, centrifuge to remove debris and then load them on the gels? Remember, i want to see ALL enzymatic acitvity...
Al Munawir, unfortunately there is no money at the moment to buy ready gels, US dollar is equivalent to 4.25 reais (Brazilian currency), plus about 100-150 dollars in taxes. No way... no funding... also, shipping takes weeks to arrive and there are no large gels, only mini...
Dear Rachel,
Simple can be good, so a crude extract would be easy to do and worth a look? In addition, bearing in mind that MMPs are secreted (although I only work in mammals, mollusca are out of my comfort zone!), what happens if you put your tissues in a very simple explant culture system, mayb for 24h and look at secreted output into the culture medium. This removes the multiple extract steps, where you may be losing, degrading, blocking your activity? It is obviously key to have a positive control. Stick with your home made gels, made fresh - they are much more consistent than the purchased ones.
Best wishes, Simon Riley
Hello Simon, thank you for your response! Unfortunately I work at a Pure Chemistry department, I have no one that could culture a few samples, which makes think difficult, and my University just started with the biology course, so nothing to do in that regard.
I think I have trypsin here somewhere, so I could you that as a positive control. Also, I think I´ll go with your suggestion of a simple extraction, I think maybe the purification protocol I´m using is too long and full of potential losses... It works for 1 and 2D electrophoresis, but for zymography doesn´t seem to work as well... :(
Maybe a crazy idea... Are they cold water molluscs? Perhaps the incubation temperature should be changed. Also, bovine/porcine collagen/gelatin may be a poor substrate Try cold water fish gelatin.
Hello everyone, after further testing I kind of got a signal... it´s not very pretty, but at least I can see some kinds of bands... Michael B LoMonaco, they are molluscs that live at about 25-35 degrees, a very ample range, since Rio de Janeiro coastal waters go from hot to cold very quickly, sometimes during the same day, but I like that idea for further studies!
Here are a few pics, if any one has an opinion... the three lanes have 60, 80 and 100 ug protein and the gel was incubated for 18 h. I was thinking of testing 40, 50 and 60 ug and varying incubation times, 16, 18 and 24 h perhaps...
Thank you!
i do zimography with mollusca samples in argentina! i could tell you how i do it!! sample extraction is very simple homegeneize with buffer, and centrifugate, load 75ug of protein, 24hrs incubation (incubation buffer with brij 35, improve a thousand time my result) dont forget to run electrophoresis at -4C.
Hello Sophia, muchas gracias por su ayuda :)
My student said 50 ug for 1 hs was the best profile she found, but I'll let her know what you said! Do you have any papers on the subject, have you published anything yet on this topic?
Saludos!
Rachel
yes, my director has plubished!
Article Correction: Endosymbiotic and Host Proteases in the Digestiv...
Hello Rachel,
Do you have tried to improve the sensitivity using Brij 35?
I recommend you first do zymography using trypsin ( or other enzyme) to fine-tune your system. Perhaps, Sophia can talk to you about your experience with Brij35
Thank you Sophia, I have downloaded the publication and will read as soon as I have time! Israel, unfortunately I don't have any money left to buy Brij, only Triton, but does it really improve sensitivity that much???
Have you tried to incubate the gels over nigth or increasing the temperature? When the protease activity is weak, these modifications of these variables can increase the protease activity
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