Casein gel zymography - (Jul/26/2013 )

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 :-) )

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.

to mdfenko: I have the same problem to solve... thanks for the great idea of soaking the normal gel in casein solution before incubation and staining. may I ask you for a protocol of the procedure? 

first, you should run the protein on a non-denaturing gel (no sds). i like to use neutral pH, non-denaturing page when possible. otherwise i use ornstein-davis page (in the past, i have posted the formulations in this forum).

if you must use sds-page, then you will have to include a renaturing step (which may be performed concurrently with the substrate soak but then may lead to inconsistent results), by soaking the gel in a non-ionic surfactant to displace the sds (eg triton x-100, nonidet p-40).

then soak in a solution of substrate.

keep in mind that the longer you soak, the more diffuse the bands will be.