SDS-PAGE Second interface - (Jul/30/2010 )
I make and run SDS-PAGs pretty frequently and I've just shown a project student the method. He casts his gels all fine and dandy and they look pretty good. However, when we run the gels (180 v ~1 hr) a second gel interface appears about half way down the resolving gel (simililar to that when forms when you layer the stacking on the resolving during casting). Some proteins stop migrating at this interface leaving a banding of high Mw proteins here, the lower Mw proteins push through and resolve fine in the rest of the gel. Does anyone have any ideas what is causing this? We will make new buffers etc but I would just like to be able to explain to the student why it's ocurring?
kriskd on Fri Jul 30 12:10:25 2010 said:
I make and run SDS-PAGs pretty frequently and I've just shown a project student the method. He casts his gels all fine and dandy and they look pretty good. However, when we run the gels (180 v ~1 hr) a second gel interface appears about half way down the resolving gel (simililar to that when forms when you layer the stacking on the resolving during casting). Some proteins stop migrating at this interface leaving a banding of high Mw proteins here, the lower Mw proteins push through and resolve fine in the rest of the gel. Does anyone have any ideas what is causing this? We will make new buffers etc but I would just like to be able to explain to the student why it's ocurring?
huh, i've never heard of that happening before. it only happens when you run the gel, not after you pour it?
sounds as though the gradient is not forming. are you sure that you are getting proper flow through the channel and proper mixing?
i second tat.. this can happen only if its a gradient gel.. if its a homogenous gel then it is virtually not possible such a phonomena!!! have heard it for the first time!!! are u sure that the pouring is proper??!!! do get back to us if the problem persists or it is troubleshooted with details... this seems to be interesting and unusual!!