Difference between Native, Reducing, and Nonreducing SDS PAGE?

[jayteeteejay]

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As the title suggests, can anyone please explain to me the differences between these three forms of SDS-PAGE? Thanks in advance

 

[eteshoe]

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):
"Native" or "non-denaturing" gel electrophoresis is run in the absence of SDS. While in SDS-PAGE the electrophoretic mobility of proteins depends primarily on their molecular mass, in native PAGE the mobility depends on both the protein's charge and its hydrodynamic size.

Take a look at the following:
http://www.ap-lab.com/native_gels.htm
https://en.wikipedia.org/wiki/Polyacrylamide_gel_electrophoresis

 

[kwu]

There is a really good video on Khan academy that goes into detail about the differences.
Here is the link:


Reducing SDS-PAGE wasn't mentioned in the video. There is a small difference between reducing and non-reducing SDS-PAGE. In reducing SDS-PAGE disulfide bonds will be disrupted.
Sometimes researchers like to compare the results between reducing and non-reducing PAGE to see if the protein contains sulfide bonds. If both tests have the same results it means that there are no disulfide bonds in the protein. If there is a different (ex: 1 band in non-reducing and 2 bands in reducing) this means that the protein contains one or more disulfide bonds.

 

[aldol16]

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):

Native PAGE is not the same as non-reducing PAGE. There are two types of SDS-PAGE... SDS is not a reducing agent - it's only a denaturant/detergent. So in reducing SDS, you add BME or another reducing agent and in non-reducing SDS, you don't add a reducing agent. Then there's also native PAGE, which doesn't have SDS at all.

 

[aldol16]

As the title suggests, can anyone please explain to me the differences between these three forms of SDS-PAGE? Thanks in advance

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.

 

[jayteeteejay]

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?

 

[aldol16]

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?

If 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.

 

[jayteeteejay]

If 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.

Got you makes sense. I appreciate it

 

[DSM_302.0]

Great thread, thanks all