gelatin zymogram - (May/19/2016 )

Jfamily on Wed May 18 13:15:16 2016 said:

I run the gelatin zymogram gel with pure human MMP9. 

After destained, the gel always has clear zone in top of the gel.

I don't know what happen on it.

I use all biorad products and follow its manual.

 

Here is an attached image and protocol.

 

1. Add Zymogram sample buffer (1 sample: 2 buffer)

2. Run a gel 

3. Wash 3X 15min in renaturation buffer (2.5% Triton X-100)

4. Wash 30 min in development buffer (5 mM Tris-HCl, pH 7.5, 20 mM NaCl, 0.5 mM CaCl2)

5. Place gel in sealable container with fresh development buffer and incubate at 37ºC for overnight (16h and 24h)

6. stain with fresh 0.5% Coomassie Blue solution for 30m 

7. Destain with MeOH:AcOH:H2O(4:1:5) for 30m-1h

 

Is there anyone who have a similar problem?

I hope to hear from you.

 

 

I don't see any clear zone - I see bands up quite high on the gel. It would pay to check your protease inhibitor concentrations and sample prep (keep things cold, use correct buffers etc.) just to make sure that the proteins aren't being degraded.