I ran a Zymogram gel and my bands are blurry and not clear to see. Why is this?
Some small proteases work better if the sample is heated for a short time before loading so the protease is denatured better and it doesn't digest the casein on its way through the gel. This may also help to improve the band sharpness.
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
What stains do you recommend for Zymogram gels?
Zymogram gels can be stained using Colloidal Blue stain or SimplyBlue SafeStain. The fixing step is not required. We recommend staining with SimplyBlue SafeStain after renaturing and developing the gel for enzyme activity. If greater contrast is needed for detecting weakly active enzymes, a 0.5% Coomassie R250 stain may be used.
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
Do protein standards run differently on a Zymogram gel compared to a regular Tris-Glycine gel?
Zymogram gels are essentially Tris-Glycine gels containing the substrate. Protein standards run based solely on the percentage of acrylamide and hence should run the same in both kinds of gels. It is quite possible though that if the standard is pre-stained, the proteins will appear a different color because of the staining (or pre-staining) of the Zymogram gels.
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
Should I boil my sample before running it on a Zymogram gel?
No, samples should not be heated or reduced so that multiunit proteases migrate as a single unit that can be renatured after electrophoresis. The sample should be one part sample and one part 2X Tris-Glycine SDS Sample buffer. Let the sample stand at room temperature for 10 minutes and then load the sample onto the gel.
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
What are the substrates for the matrix metalloproteases (MMPs)?
The substrates for the matrix metalloproteases (MMPs) are:
*MMP-1: tissue collagenase: collagen 1, 2, 3, 4, 6, & 10
*MMP-2: gelatinase: gelatins, collagens 4, 5, & 7
*MMP-3: stromleysin-2: casein, fibronectin, laminin, elastin
*MMP-7: matrilysin: casein
*MMP-8: neutrophil collagenase: collagen
*MMP-9: type 4 collagenase: gelatin
*MMP-10: stromelysin-2: casein
*MMP-12: metalloelastase: elastin
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
