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Position: Discussion forums > Bioscience biotechniques discussion forums > Proteomics and protein biochemistry forum
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protein aggregation in SDS PAGE
Posted by: angelaniki (IP Hidden, New member, 2)
Date: October 27, 2005 08:03AM
I've ran so many gels and this is the first time it doesn't work at all!!
All the samples got stocked at the bottom of stacking gel (the head of separating gel), so there are only thick bands at the separating gel head, very few separated bands (only 1 or 2). The most weird thing is that even markers got stocked! Everyone in the lab use the same markers and it works perfectly, so that must be the problem of the gel itself! And then i tried my colleagues buffers, recipe....everything, and same problem again! I just couldn't solve it nor couldn't i explain!! I rinsed the butanol (or ethanol) w/dH2O many times b4 layering stackig gel, and samples are fully defrozed at room temp, so not the SDS precipitate, what elase could it be??? Should i stain gel longer(i do 5min)?? Please help! Thanks!
Re: protein aggregation in SDS PAGE
Posted by: kum81 (IP Hidden, Junior member, 10)
Date: February 8, 2006 06:17AM
Hi it seems there is some problem with your resolving -stacking interphase.or the resolving gel hi First of all make all the reagents fresh carefully adjust the pH. make a fresh set of sample buffer also. insted of butanol simply overlay the seperating gel eith distilled water. make sure that u denature the proteins properly and spin before loading stain with coomassie stain for at least 30 min hope this helps you
Re: protein aggregation in SDS PAGE
Posted by: mitolab (IP Hidden, Senior member, 89)
Date: February 15, 2006 05:45PM
If you are using home made polyacrylamid gel, be sure to prepare fresh acrylamide stock buffer every one month. You may consider to switch to commercial mini gel to save your precious time.
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