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Casein gel zymography

gel zymography casein proteases

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#1 bioseb

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Posted 26 July 2013 - 12:57 AM

Hello everybody,

I have applied gel zymography to visualize an activated protease after electrophoretic separation. For that purpose, 0.1% casein was incorporated into the 7.5% polyacrylamide gel as the protease substrate. Now I was wondering whether one should consider an influence of the casein on the general protein separation. Is it possible that the gel becomes 'densified' to some degree by incorporation of the abovementioned amount of casein?

I had this idea after having observed that the active protease accumulated at the upper gel edge. In contrast, the enzyme was separated as usual in common SDS-PAGE with a 12% SDS polyacrylamide gel. Of course I should also consider the initial non-reducing conditions in gel zymography. But nevertheless, I wonder whether the casein might have any effects on separation, too.

Thanks in advance. I would be glad to get some ideas for my discussion of these results.

Bioseb


(Sorry in case my English is not completely correct :-) )

#2 mdfenko

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Posted 26 July 2013 - 03:31 AM

the casein should have no significant effect on the running of the gel. however, it will migrate through and out of the gel so you need to include an equal concentration of casein in the upper reservoir (-) buffer.

alternatively, you can run normal native page and briefly (~15-20 minutes) soak the gel in a solution of casein (as you would with a stain) in a buffer which will allow for proteolytic activity.
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