MMP activity in zymography - proteinase activity in SDS (Oct/21/2005 )
Needing some help with gelatin zymography. How do MMP's maintain their proteolytic activity during gelatin zymography while in the presence of 10% SDS?? Any thoughts?
-schrammie-
QUOTE (schrammie @ Oct 21 2005, 08:11 AM)
Needing some help with gelatin zymography. How do MMP's maintain their proteolytic activity during gelatin zymography while in the presence of 10% SDS?? Any thoughts?
I think MMP is coated with SDS to prevent its proteolytic activity when the gel is running or else the MMP will disgest the gelatin while it is electrophorzed!! So that you will not have a band after washing with triton x and developed with coomassie blue. You may see a light swear on the gel.
-Minnie Mouse-
QUOTE (Minnie Mouse @ Oct 23 2005, 04:38 PM)
QUOTE (schrammie @ Oct 21 2005, 08:11 AM)
Needing some help with gelatin zymography. How do MMP's maintain their proteolytic activity during gelatin zymography while in the presence of 10% SDS?? Any thoughts?
I think MMP is coated with SDS to prevent its proteolytic activity when the gel is running or else the MMP will disgest the gelatin while it is electrophorzed!! So that you will not have a band after washing with triton x and developed with coomassie blue. You may see a light swear on the gel.
After running the gel it is washed in triton which removes the SDS and allows the enzyme to refold and regain activity. The refolding is not complete which can be seen by the bands made by the "inactive" MMP ( the tail that is normally cleaved to activate the ezyme remains denatured which also activates the enzyme). During the measurement of proteolytic activity the MMP is NOT in the presence of SDS.
-ajames-
QUOTE (ajames @ Oct 23 2005, 06:28 PM)
QUOTE (Minnie Mouse @ Oct 23 2005, 04:38 PM)
QUOTE (schrammie @ Oct 21 2005, 08:11 AM)
Needing some help with gelatin zymography. How do MMP's maintain their proteolytic activity during gelatin zymography while in the presence of 10% SDS?? Any thoughts?
I think MMP is coated with SDS to prevent its proteolytic activity when the gel is running or else the MMP will disgest the gelatin while it is electrophorzed!! So that you will not have a band after washing with triton x and developed with coomassie blue. You may see a light swear on the gel.
After running the gel it is washed in triton which removes the SDS and allows the enzyme to refold and regain activity. The refolding is not complete which can be seen by the bands made by the "inactive" MMP ( the tail that is normally cleaved to activate the ezyme remains denatured which also activates the enzyme). During the measurement of proteolytic activity the MMP is NOT in the presence of SDS.
Thanks for the useful info!!!
-schrammie-