zymography assay - (Sep/24/2008 )
hi.guys
I am going to do the zymography assay.Could you give me some suggest.
Firstly,what do you use as a internal control to ensure that the MMPs are screted by the same number of cells?
Secondly,if we use SDS-PAGE, the MMP maybe denatured and lose their activity,but if we use native-page, we may not be able to recogonize MMP by molecular weight
what to do with the problems? many thanks!
-valind-
QUOTE (valind @ Sep 24 2008, 11:39 AM)
hi.guys
I am going to do the zymography assay.Could you give me some suggest.
Firstly,what do you use as a internal control to ensure that the MMPs are screted by the same number of cells?
Secondly,if we use SDS-PAGE, the MMP maybe denatured and lose their activity,but if we use native-page, we may not be able to recogonize MMP by molecular weight
what to do with the problems? many thanks!
I am going to do the zymography assay.Could you give me some suggest.
Firstly,what do you use as a internal control to ensure that the MMPs are screted by the same number of cells?
Secondly,if we use SDS-PAGE, the MMP maybe denatured and lose their activity,but if we use native-page, we may not be able to recogonize MMP by molecular weight
what to do with the problems? many thanks!
I always do a DNA determination on the cells (Cyquant), so then you know if you have about the same amount of cells.
What do you mean with SDS-Page, just running on the coated-zymogram gel? You need to run the samples on gel in a sample buffer with SDS but without any other reducing agents and you must not boil them. However, due to the SDS and running on the gel, you do get some kind of denaturing (hard to explain, may be google can tell you more). Due to this, you can also see the fragments of the inactive enzymes (MMP's are sectreted in a pro-form and need to be cleaved for activation). So, if I run eg on a gelatin coated gel, I do see two fragments for eg MMP2. MMP 2 pro-form is 72 kDa, active (cleaved) form is 68 kDa.
-aspergillie-