Hi Miriana,

I have lots of experience with tPA zymography, but it was on casein-plasminogen-agarose gel overlay.

I assume that you are making the casein-plasminogen gels yourself. If so, the acrylamide, BIS and TEMED do degrade. Make new gel reagents and see what happens.

Hi Steingrimur and thanks for your reply.

Indeed I am making the gel myself. Unfortunately I have tried already to change all the buffers, I have also autoclaved bottles and water. If you have any other suggestions please let me know :)

Hi Miriana, I also assume that you are storing casein at -20 and Plg at -80.

What about the acrylamide, BIS and Ammonium Persulfate solutions? Do you store them or do you make them up fresh?

Do your gels look uniformly blue after the staining/destaining? Or is the dye front not staining with coomassie?

Hi Steingrimur, and thank you for your comment.

I am storing casein at -20 and Plg at -80. Both of them were aliquoted in November 2015.

Can it be that the substrates are no more active?

Regarding the solutions for the casting of the gels, I have tried with both fresh and the ones stored at +4C, no difference.

When I stain and destain the gel looks very uniform.

Thanks again,

Miriana

Hi Miriana, try running trypsin on the casein gel along with active plasmin. If you don't see clearing hen there is something wrong with the gel.

If you see trypsin and plasmin, then there is something wrong with the Plg. 

Dear

Please check the following publication to find another interesting informations: Use of Gel Zymography to Examine Matrix Metalloproteinase (Gelatinase) Expression in Brain Tissue or in Primary Glial Cultures. Harald Frankowski et al. 2012. Methods Mol Biol. 2012; 814: 221–233. Directlink: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3670093/

Hoping to be helpful

Marius