:: Search forums :: Top Users :: Reagent
Position: Discussion forums > Bioscience biotechniques discussion forums > Proteomics and protein biochemistry forum
|
Re: Why does butanol creat two an extra layer of gel above seperating gel?
Posted by: femmeauburn (IP Hidden, Advanced member, 115)
Date: July 7, 2006 10:39AM
Wait I too am confused. The stacking gel should be poured after the separating (resolving) gel has completely polymerized and the overlay solution (water, butanol, etc) has been removed. So how does the expelled water find its way into the base of the stacking gel? I think it could only happen if one did not allow enough time for the resolving gel to polymerize or if the overlay was not removed. Also as olivialei states that it had already formed before the stacking gel, this cannot be what is happening. I think instead her problem was mixing of the resolving gel and the butanol at the overlay surface.
Re: Why does butanol creat two an extra layer of gel above seperating gel?
Posted by: olivialei (IP Hidden, Junior member, 13)
Date: July 7, 2006 11:57AM
I think I agree with femmeauburn. But another interesting thing I noticed is that my rainbow marker couldn't get seperated in that extra layer of gel. If it is the mixture of resolving gel and butanol, should it seperate proteins too? Could you tell me that advantages of using water saturated butanol over just water as the overlay solution?
Re: Why does butanol creat two an extra layer of gel above seperating gel?
Posted by: femmeauburn (IP Hidden, Advanced member, 115)
Date: July 7, 2006 03:44PM
I can think of two reasons why the marker could not separate in the extra gel layer. One, the concentration of polyacrylmide may be too low due to its mixing with the butanol; at least 4% is typically required for separation. Two, the gap between the extra gel layer and the regular separating gel may interfere with electrophoreis if large enough. No current, hence no separation. I do not know of any advantages of butanol; we have always used water. Remember to remove the butanol promptly after polymerization, leaving it on too long can result in poor well formation due to shrinkage of the resolving gel. This shrinkage cannot happen with a water overlay.
|
We are moving ... Please post to our new community forums
