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jayteeteejay
As the title suggests, can anyone please explain to me the differences between these three forms of SDS-PAGE? Thanks in advance
I was gonna write a long answer but I'm don't really feel like typing out too much (also native=non-reducing PAGE, SDS is the reducing detergent so there are just two types of PAGEs):
As the title suggests, can anyone please explain to me the differences between these three forms of SDS-PAGE? Thanks in advance
Great answer! Just to clarify, if you have heterodimers under SDS-PAGE then they will appear on separate bands whereas native PAGE the heterdimer will appear as a single band?You should seek to understand this in two parts: 1) what each of these are technically and 2) what each of these do. So with regard to (1), you can run either reducing or non-reducing SDS PAGE. SDS is a denaturant and it coats the protein with charge so that intrinsic charge no longer matters and the proteins migrate according to size. But SDS does not break disulfide bonds under non-reducing conditions and if your protein has this, it could mess up your gel because then 3D shape will matter and the proteins won't just be moving by size. That's why the majority of gels are run under reducing conditions (reducing SDS), which disrupt the disulfide bonds and makes the proteins truly migrate due to size. These two methods offer a good way of telling whether your protein 1) has disulfides (different gel pattern) and 2) whether those disulfides link up subunits/domains (getting 1 band in non-reducing and then 2 bands of comparable smaller size in reducing SDS).
Finally, native PAGE is run in the absence of SDS and since SDS is a denaturant, the protein runs in its native, or folded, form. This is not very useful for quantitative purposes but is very useful if you're trying to figure out if your protein is globular or has a lot of beta strands. Sheets will present a larger surface area and thus draw more drag whereas globular proteins will migrate quickly. Experimentally, you would run one of these with several other known globular and beta stranded proteins so that you can compare.
Great answer! Just to clarify, if you have heterodimers under SDS-PAGE then they will appear on separate bands whereas native PAGE the heterdimer will appear as a single band?
Got you makes sense. I appreciate itIf you have heterodimers linked via non-disulfide interactions, then yes, they will appear in separate bands on SDS-PAGE and single band in native PAGE given that the interactions provided by SDS are enough to break up those dimers. There are very stable dimers that can't be broken apart and those require more teasing. But in general terms, yes. Even homodimers would be distinguishable - you look for a single band under SDS conditions that is lighter than the band that appears in native PAGE.
Now, if those dimers were linked by disulfides, you would need reducing PAGE to tease them apart.