4 replies to this topic

[Mieke]

Hello everybody,

I am having a problem with doing gel-zymography for quite a while now. The samples don't seem to migrate properly when running my gel. Once they leave the stacking gel, they just stay put and don't migrate further. The ladder does migrate just fine and the loading buffer also moves to the bottom.
Does anyone know a solution for this? (I know people who use the exact same protocol and don't have this problem!)

Many thanks in advance!

Mieke

[mdfenko]

it sounds like your protein is aggregated.

you used sds-page? if so then you may have overboiled your sample. try boiling for 5 minutes or less or heat at 65-70C for 10-20 minutes.

[Mieke]

mdfenko, on 06 January 2011 - 01:34 PM, said:

it sounds like your protein is aggregated.

you used sds-page? if so then you may have overboiled your sample. try boiling for 5 minutes or less or heat at 65-70C for 10-20 minutes.

No I did a gel zymography with a 15% gelatin gel, so I don't boil my samples. As a result I see all my bands I want to see but they are packed together on top of my gel.

[mdfenko]

they may just be large proteins. try a lower percentage, less restrictive gel.

[BradH]

Hi Mieke,

I am having what sounds like the same problem as you. Did you ever find a solution?

Thanks,
Brad