Thermo Fisher has commercial kit, search kinase enrichment kit (ATP or GTP)

Hi Bassel, this is not going to be easy.

Specific metalloproteinase activities are hard to pin down because many of them hydrolyze the same substrates. And since they generally do not form covalent intermediates like serine and cysteine proteinases, specific inhibitors are sparse.

So as far as I know, the only way I know of to identify metalloproteinases based on their activities is to look at how well they cleave quenched fluorescent peptide substrates.

There are companies that  sell FRET-based peptide substrates for metalloproteinases:

http://www.korambiotech.com/upload/bbs/2/Fret%20Brochure%20new%20version%20111607.pdf

http://shop.bachem.com/peptides-and-biochemicals/matrix-metalloproteinase-mmp-inhibitors-and-substrates.html

@Bin Fang i need a kit for metallo protease and kinake

@ Steingrimur Stefansson :

thank your for your answer, and sorry for my late reply you are right, because metallo protease do not make a covalent bond, one needs a photo cross linker, which makes a covalent bond after metall chelating, which enables its detection and identification. However, it is commercially not available and should be sythesized, which is not possible in our situation. this method makes it possible to identify metalloproteases which is important in this case for me, is it possible to achieve this with FRET?

Hi Bassel, if you have antibodies to your metalloproteinases of interest, you can try what this group did: 

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3620676/pdf/1756-6606-6-14.pdf

Basically, they coated ELISA wells with an anti-MMP Ab to capture the proteinase and then came in with a FRET substrate and measured the fluorescence.

Hi Steingrimur,

it is different a little bit in my case, because my aim is to identify (as many as possible) metalloprotease in aspergillus cell culture supernatant. most of aspergillus proteases are uncharacterized and i do not know exactly which proteases are secreted. 

Have you done gelatin zymography +/- EDTA to get an idea of how many MPs there are in the supernatant?

Hi Steingrimur,

no i did not, i performed a general protease acitivity assay +/- EDTA. 50% of the activity in the sample is reduced after using EDTA

do you think i can perform gelatin zymography and then cut the gel bands and digest them with trypsin to identify them? 

Hi Bassel, how do you measure proteinase activity? Azocasein or other nonspecific protein substrate?

If you are using nonspecific protein substrate, then it is a good sign that you are losing 50% of the activity in the supernatant with EDTA.

It will be tricky to perform trypsin digestion on a gelatin zymogam. For one thing there are plenty of gelatin fragments around to complicate analysis. Secondly, gelatin gels can detect very low concentrations of proteinases, so you might not have enough enzyme to analyze.

Just saw your answer that the proteinases are not characterized. So there is no really good way to profile the proteinases unless you use some kind of non- specific FRET substrate, minus and plus a general MP inhibitor like O-phenanthroline and EDTA. If they are MMP like, they will cleave peptides which have a proline at S3, and many companies sell the small FRET substrates like AnaSpec and they are cheap. If you can predict to some extent the type of metalloproteinases in your organism, that would help as well. Are they MMP like! for example? Would they be just metzincins, or would others be present? If you know the types of MPs that might be present, it would be easier to purchase the correct types of substrates. 

I do think zymography and cutting out the bands to characterize is a great idea, BTW. You can try to purify the higher MW bands from the gelatin fragments, perhaps by running small spin columns. The high MW material will elute and low MW material, ie 1Kd or less will be retained. or simple dialysis with different MW cut offs. You can also try affinity columns like gelatin sepharose, and benzamidine columns and then mass spec what is retained and then eluted from the column. 

Steingrimmer,

i measured the protease activity using azocasein. 

Macia, thank you for you reply. i think it is a good idea to purify them from gelatine using a spin column. but as you know gelatine is a substrate for some metalloproteinases and not for all of them, so i think gelatine zymography will deliver a partial info about the metalloproteinase in the sample, an i need also to apply casein and collagen zymography?

Hi Bassel, I think the first things you need to identify are how many MPs you have in your sup. and what is their MW. Zymography is the quickest way to determine those parameters. Plus, you can also do proteinase inhibitor profiling using zymography. 

If you find an inhibitor for the MPs, then you can start looking for affinity resins.

Since you know that the proteinases cleave casein, you can incorporate that in the gel instead of geatin.

Hi Steingrimmer, 

that's it, i will start with casein. do you have a protocol for it?

Hey Bassel, there are plenty of protocols out there. You will have to check them out and see which one fits your situation the best.

http://scholar.google.com/scholar?q=%22casein+zymography%22+metalloproteinase&btnG=&hl=en&as_sdt=0%2C21

Hi Steingrimmer, 

thank you so much for your help, 

Actually, it is Steingrimur, a fine Icelandic name, but you are welcome.

ah, sorry :D 

 Hi,

Sigma's non-specific protease activity assay may be used as a standardized procedure to determine the activity of proteases.

http://www.jove.com/video/899/sigma-s-non-specific-protease-activity-assay-casein-as-a-substrate