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Position: Discussion forums > Bioscience biotechniques discussion forums > Proteomics and protein biochemistry forum
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Why does butanol creat two an extra layer of gel above seperating gel?
Posted by: olivialei (IP Hidden, Junior member, 13)
Date: April 2, 2006 09:31PM
I have a question related to SDS-PAGE. After pouring the seperating gel, I added about 100 ul water saturated butanol above it. When it polarmizes, I noticed that there was an extra layer of gel above the seperating one. It is about 3 mm thick. My samples were not separated until they entered seperating gel. I was wondering what is that extra layer of gel. How did it happen? If anybody knows the explanation, please let me know. Thank you so much!
Title correction: Why does butanol create an extra layer of gel above seperating gel?
Posted by: olivialei (IP Hidden, Junior member, 13)
Date: April 2, 2006 09:39PM
I'm sorry about the typos in the title of my last posted message.
Re: Title correction: Why does butanol create an extra layer of gel above seperating gel?
Posted by: Noor (IP Hidden, New member, 2)
Date: May 2, 2006 03:56AM
I think you should clear off butanol before you pour your stacking gel. You could also just use water.
Re: Why does butanol creat two an extra layer of gel above seperating gel?
Posted by: femmeauburn (IP Hidden, Advanced member, 115)
Date: June 6, 2006 04:12PM
what could have happened is that the butanol mixed with the separating gel below the surface of the gel. this would create a gap within the gel (or the extra layer you see) as not enough gel would be in this butanol layer to polymerize. to avoid this be certain to gently add the butanol over the gel. adding it with too much pressure can force the butanol into the gel.
Re: Why does butanol creat two an extra layer of gel above seperating gel?
Posted by: olivialei (IP Hidden, Junior member, 13)
Date: June 7, 2006 08:20PM
Thank you, femmeauburn. I think your explanation might be correct since that extra layer of gel became thinner or almost invisible when I added less butanol. It seems to me that water has less possibity to creat this kind of problem.
Re: Why does butanol creat two an extra layer of gel above seperating gel?
Posted by: femmeauburn (IP Hidden, Advanced member, 115)
Date: June 8, 2006 10:21AM
We usually use water to overlay the gel. I haven't add any problems with mixing.
Re: Why does butanol creat two an extra layer of gel above seperating gel?
Posted by: ianbirmingham (IP Hidden, New member, 7)
Date: July 5, 2006 10:02AM
When acrylamide polymerises water is produced- thus my isopropanol overlay becomes water saturated during polymerisation. Now, if the acrylamide isnt allowed to fully polymerise (set but not fully set) prior to layering the stack gel ontop, ie through rushing, it is possible to produce a three tier gel. 1. Separating gel- 2. water/stack gel ie very low percentage stack- 3. stack gel. This is my explanation of what I think you are experiencing.
Re: Why does butanol creat two an extra layer of gel above seperating gel?
Posted by: olivialei (IP Hidden, Junior member, 13)
Date: July 5, 2006 10:43AM
Thanks for your explanation on my question. However, I don't think I fully understand it. According to your explanation, that extra layer of gel should disappear if I give enough time to the polymerization of seperating gel. I'm not sure if it is true. By the way, the butanol I use is already water saturated. It should not affect anything even though polymerization of acrylamide and bisacrylamide can produce water.
Re: Why does butanol creat two an extra layer of gel above seperating gel?
Posted by: ianbirmingham (IP Hidden, New member, 7)
Date: July 6, 2006 03:20AM
What happens, I think, is that water keeps on being expelled from the separating gel as it completes its polymerisation, which finds its way into the base of the stack gel- resulting in the optical density of the base of the stack gel being different to the stack and the separating gel.
Re: Why does butanol creat two an extra layer of gel above seperating gel?
Posted by: olivialei (IP Hidden, Junior member, 13)
Date: July 6, 2006 10:17AM
I'm not completely convinced because that extra layer was already formed before the stacking gel.
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