the relfoded sample was prepared in SDS containing buffer and hetaed then load on PAGE gel while the same sample was prepared in buffer without SDS but DTT was present and without heating load on SDS PAGE. what was the basic principle to observe refolding by this way?
The idea is to check if disulfide "S-S" bonds have formed during refolding. You run a protein after heating on SDS-PAGE, with or without reducing agent (DTT or 2-mercaptoethanol). The reducing agent breaks S-S bonds. If S-S bonds are present, then the two different samples will migrate at different positions (usually). If no S-S bonds were formed then the two samples will migrate at the same position. We call this analysis boiled-reduced versus boiled-nonreduced SDS-PAGE. It may tell you if you have S-S bonds but will not tell you if your protein is correctly folded. Also, these kinds of gels are sometimes called "denatured" because your sample gets unfolded by the SDS and heat.
Proteins migrates proportionally to their molecular weight on denatured gels (boiled-reduced) because hundreds of copies of negatively charged SDS uniformly coat the extended molecule. If you you don't heat your sample, and lower the amount of SDS in your loading buffer and gel ...so that you don't unfold your protein... you can run a "native" gel. A correctly folded protein usually gives a single tight band on native SDS-PAGE. An unfolded protein will usually give a smear from the top to the bottom of the lane. I often run native gel on FPLC fractions to find my refolded protein. Several companies sell precast gels and loading buffers for native page.
Good luck
The idea is to check if disulfide "S-S" bonds have formed during refolding. You run a protein after heating on SDS-PAGE, with or without reducing agent (DTT or 2-mercaptoethanol). The reducing agent breaks S-S bonds. If S-S bonds are present, then the two different samples will migrate at different positions (usually). If no S-S bonds were formed then the two samples will migrate at the same position. We call this analysis boiled-reduced versus boiled-nonreduced SDS-PAGE. It may tell you if you have S-S bonds but will not tell you if your protein is correctly folded. Also, these kinds of gels are sometimes called "denatured" because your sample gets unfolded by the SDS and heat.
Proteins migrates proportionally to their molecular weight on denatured gels (boiled-reduced) because hundreds of copies of negatively charged SDS uniformly coat the extended molecule. If you you don't heat your sample, and lower the amount of SDS in your loading buffer and gel ...so that you don't unfold your protein... you can run a "native" gel. A correctly folded protein usually gives a single tight band on native SDS-PAGE. An unfolded protein will usually give a smear from the top to the bottom of the lane. I often run native gel on FPLC fractions to find my refolded protein. Several companies sell precast gels and loading buffers for native page.
Good luck
Thankyou sir for such a nice description of my problem. i think i get my answer.
well explained Chris, now I have to show that denatured and refolded protein migrate different position. CD analysis showed that protein has refolded .refolded protein have only one Cys residue so no way of S-S bond. so how should i use sample buffer to show denatured n refolded bands at diff position (due to diff migration)
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