hello Alessandro. Would you like a protocol to zymogram from protein obtained by a buffer extraction or in situ, that is, to detect activity of mmps in the sections on the slides?

The in Vivo zymography I saw in one paper by BD Crawford et al, attached fret substrates to some kind of matrix. This is not hard to do. But for your purposes, you can just add the fret substrates to the brain extract or slices and then measure fluorescence. We use FRET substrates with excitation and emission of 485/530nm. So I would take MMP buffer, we use 50mM tris, pH8 , 10 mM cacl2, and 150mM NaCl. The substrates, are added from DMSO stocks, to 10uM final. And the brain extract, which would be a slurry, is added to start the reaction. The brain extract can be prepared in the same MMP buffer. Just make a suspension of about 1mg of material in 1 ml. Then vortex, and add the suspension to the 100ul of substrate/ buffer, and measure the increase in fluorescence.

For the brain slices, you can incubate the slice with he substrates too. If they are on a slide, then I would add buffer/ substrate, and use a slide reader to measure the change in fluorescence. You can also probably attach substrates to a gel and add to the slide. I can give you a design for substrates that should work, but that wouldw be labor intensive.

If you want certain placement of the substrates, ie to regions in the brain slice, then I would use an ab attached to he fret substrates. This is not hard to do either. You can contact me via researchgate for more info. I can send substrates for you to try.

If you need to quantify different MMP activities, or get exact numbers, you can use the same substratEs, but would have to set up a collaboration with colleagues of mine, and I don't know if they have time to do things. Brut it doesn't hurt to ask.

The substrates we have are somewhat selective for MMP2, MMP9, and MMP13. We also have one that is a broad spectrum MMP substrate.

I have did protein zymograms on non-denaturating PAAG protein gel. You simply extract proteins from biological material (yeasts in my case), do not denaturate them, add proteinase inhibitors to ensure stability of the proteins and then do PAAG electrophoresis in +4 gradC and then do specific staining (it works for dehydrogenases, might work for phosphotases and peroxidases).

I am afraid, it won't work that way with proteinase since one should inhibit their activity during protein extraction.

Display Settings:AbstractSend to:

Methods Mol Biol. 2010;622:271-7. doi: 10.1007/978-1-60327-299-5_17.

In situ zymography.

George SJ, Johnson JL.

Source

Bristol Heart Institute, Bristol Royal Infirmary, University of Bristol, Bristol, UK.

Abstract

In situ zymography is a unique laboratory technique that enables the localisation of matrix-degrading metalloproteinase (MMP) activity in histological sections. Frozen sections are placed on glass slides coated with fluorescently labelled matrix proteins. After incubation MMP activity can be observed as black holes in the fluorescent background due to proteolysis of the matrix protein. Alternatively frozen sections can be incubated with matrix proteins conjugated to quenched fluorescein. Proteolysis of the substrate by MMPs leads to the release of fluorescence. This technique can be combined with immunohistochemistry to enable co-location of proteins such as cell type markers or other proteins of interest. Additionally, this technique can be adapted for use with cell cultures, permitting precise location of MMP activity within cells, time-lapse analysis of MMP activity and analysis of MMP activity in migrating cells.

I would also like to know the protocol, Jose, for the in situ zymography. I would imagine that FRET peptide substrates could replace the matrix protein- fluorescein type substrates.

Hi Jose Gomes, I would appreciate both since I have no experience in detecting protease activity and it would be interesting to learn both methods...Thank in advance!

Thank you very much for the detailed help.

Thanks were in particular for Marcia Moss, of course!!

Hi Alessandro,

I would like to refer you to a colleague of mine, Jennifer Vandooren who has a lot of experience with zymography and has written a nice Nature methods on the subject.

have a look at http://www.nature.com/nmeth/journal/v10/n3/full/nmeth.2371.html

Thanks a lot Julie!! I'll ask your colleague if she can send me a copy of the paper...Thanks again...

Hi Alessandro,

I believe that the major updates and outstanding methods are described in:

Zymography methods for visualizing hydrolytic enzymes. Vandooren J, Geurts N, Martens E, Van den Steen PE, Opdenakker G. Nat Methods. 2013 Mar;10(3):211-20. doi: 10.1038/nmeth.2371. Review.

good luck and reading

Ferdinando

Alessandro,

A very simple method used for rapid screening of serine based hydrolytic enzymes can be found in;

Use of coomassie blue-polyurethane interactions in screening of polyurethanase proteins and polyurethanolytic microorganisms, Howard, G. T. and Hilliard, N. P., International Biodeterioration and Biodegradation, 43, 23-30, 1999.

The method involves use of the familiar Coomassie-blue staining methods available in most labs. Hope this helps.

Newt

Hi Alessandro. well, do you knew that the mmps are secreted by cells in a Pro MMP form ( for the matrix metaloproteinases) or may remained attached to cell membrane (for the MT-MMP) right!. In both case they need to be activated by other mmp or other molecules to degrade its specific substrates. Then, when the zymograms are obtained from a tissue homogenization using a buffer the MMPs in the Pro form also are activated by method and appear on gel closed to active form. This method is very very sensible and you can obtain a result using only 0,25ug of total protein from the sample. The careful in this method depends the aims that you want to reach. If your interest is only detect the presence of mmp in a tissue, for example, along of different phases during the morphogenesis the careful is basic however if the interest is quantified the activity of mmp the careful during the procedures of method must to be doubled specially during the pipetting of each sample. For this measurement it is necessary to have a calibrate curve with a mmp purified to compare with that obtained in the samples. Because of great sensibility of this method to quantify the mmp activity is also prudent to do dilution of the sample to assure that the activity measured is true. Finally although exist the basic protocols to perform a zymogram or a zymogram in situ always is important test they for your tissue of study. Alessandro, if you want more detailed send a mail me by [email protected] I will appreciate help you with my modest experience.

Marcia Moss. hello. I do not know the FRET method, is it based on protein fluorescent substrates too, like in situ zymography? could you talk about the FRET? thanks

The substrates we have are peptides that have the donor and acceptor fluorophores pairs. When the peptides get cleaved, the fluorescence increases, because the fluorophores pairs are no longer close to each other. The substrates we have were used with extracts from cells, cell media, and tissue homogenates as well as urine, plasma, serum, saliva, and endometriotic fluids. They have not been used with slides yet, but can I believe because protein substrates can be used. But our substrates are more specific for individual MMPs and ADAM family members. So I did not have access to the nature methods paper, but from the one figure I could see, our substrates could be used. If anyone can send me the paper, that would be great. And if anyone wants to try the substrates, that would be great too.