Zymograms - Does anybody have a protocol? (Feb/23/2005 )

Hi. I am trying to determinate enzyme activity by zymogram ... I never worked with this technique. Please..Can anybody help me?


Fredy

I believe you can isolate zymograms by percoll gradient

Simon

I just had to do this recently (first time). This protocol is quite good.

http://www.chemicon.com/techsupp/Protocol/...nzymography.asp

Zymograms are run the same as SDS-PAGE but you have to make your own gel and add either gelatin or casein depending on the protein you are trying to detect.

Gelatin is for MMP-2/9 activity. I was using stem cells and brain tissue so I also incubated my lysis buffer + protein in Gelatin Sepharose 4B.

Unfortunately I do not know too much about Casein zymography so I hope your looking at MMP-2/9.

Hope this helps!

I've used a protocol very similar to the one on chemicon.com (link listed in previous message) and it worked great. The one I'm using is from Invitrogen - it's the protocol in their Novex Pre-Cast gel instruction booklet.


I'm wondering if anyone knows how to make their own zymogram gels as opposed to buying the precast ones. Does anyone have a protocol/recipe?

Thanks!

Depending on the crosslinker (ratio of Acrylamide:Bis solution) it may differ. Bisacrylamide serves as a crosslinker. The amount of bis therefore determines the porosity of the resulting gel. The more bis you use in a gel the smaller are the pores in the gel.

- 37.5:1 for the separation of high molecular weight proteins
- 29:1 for sequencing gels and the separation of proteins
- 19:1 Optimal for sequencing gels and also for the separation
of peptides

In this example I'll use 37.5:1 and a 7.5% acrylamide gel.

You need a resolving gel that will fill most of your plate and a stacking gel which is only a filled a bit lower than the combs will fit. (I'm hoping your using vertical gels). If you make 20 mLs of resolving and 10mLs of your stacking you would get a recipe that looks like this.

Stock Solution of Acrylamide:Bis (If you buy separately)
Acrylamide 30g
Bis 0.8g
QS w/dH20 to 125mL

Make a solution of whatever % of gelatin you want your gel and add it to the resolving gel.

Resolving Gel (20mLs):
Acrylamide Solution 5mL
1.5 Tris pH 8.8 5mL
10% SDS 200uL
H2O ~8.6mL
10% APS 200ul
TEMED 20ul
0.15% Gelatin mixture 1mL

Stacking Gel
Acrylamide 1.275ml
1.0M Tris pH 6.8 1.3ml
10% SDS 100ul
H2O 7.25ml
10% APS 100ul
TEMED 10ul

Once you add TEMED (do it last) to these it will solidify quite quickly so you need to cast the gel very fast.

It's a big pain in the ass to figure out your own ratios depending on what you are trying to do but it may or may not be worth it depending on how much $ you have.

Hope this helps.

Thanks a lot!

Hi! Thanks for the gel recipe. I just have one more question. What type of gelatin do you use and where do you buy it?

Thanks!