laczlacylaci

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Native-page: everything in-tact, separated by size and charge or shape.
ie. If we had a heterotrimer, we only would see one band.
SDS-page non-reducing(w/o B-ME): S-S are intact but protein is denatured
ie. If we had a heterotrimer, we would only see one band.
SDS-page reducing (w/ B-ME): S-S become reduced protein is still dentured.
ie. If we had a heterotrimer, we would see 3 separate bands.

From this picture, we can say that the protein was a heterodimer (or whatever 'mer' in this case, just it can create 2 different KDa of subunits)





In this picture, I notice that the non-reducing detected higher MW proteins than reducing. Looking at lane 5, what can we deduce? If it were to be a homodimer, then I think I would only see one band at around 7kDa and not 18.4.

I guess what is the main difference between native-page and SDS-page non-reduced?
The only thing I can think of for now is that if you run a heterodimer under both.
If you see one band for native-page and 2 bands for SDS-page, then the dimers weren't connected by a disulfide bridge.
 
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aldol16

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I guess what is the main difference between native-page and SDS-page non-reduced?
In a native gel, you don't denature the protein at all. So the 3D shape becomes important in determining how the protein migrates. In a non-reducing SDS-PAGE, you still denature the protein - you just leave the disulfide bridges intact. So the protein will be mostly denatured and if it has disulfides, those will convey some 3D structure but very minimal compared to native gels.

If a protein has multiple subunits that are held together by disulfides, then those subunits will come apart under reducing SDS but not under non-reducing SDS. In other words, you'll see low-weight units under reducing conditions but heavier fragments under non-reducing SDS. If you ran a native gel on the same protein, you would see either apparently "lighter" or apparently "heavier" fragments than non-reducing SDS, depending on whether the protein is globular, predominantly beta-sheeted, etc.

Keep in mind that disulfides can exist independently of subunits. So disulfides could be intra-unit and not function to hold multiple subunits together but rather to hold bits and pieces of one subunit together to convey local 3D topology.
 

akimhaneul

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If you ran a native gel on the same protein, you would see either apparently "lighter" or apparently "heavier" fragments than non-reducing SDS, depending on whether the protein is globular, predominantly beta-sheeted, etc.
Hello, do you mind explaining this more? I don't quite understand. Why would it be either lighter or heavier?
Also for that bottom gel titled non reducing and reducing, I notice that some of the lanes in non reducing gel is lower than the ones in reducing. Didn't you say that bands in reducing gels should be lower in weight?
 
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laczlacylaci

laczlacylaci

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Hello, do you mind explaining this more? I don't quite understand. Why would it be either lighter or heavier?
http://forums.studentdoctor.net/threads/native-page-vs-non-reducing.1194413/
I think there is a similar question going on in an older thread.

upload_2016-8-28_13-38-18.png
I think native-page just gives more info on protein strucutre (more coiled/tightly folded->will travel/migrate further)
I am not so sure what aldol16 meant by heavier or lighter, like band color intensity?

I wonder why 'supercoiled' DNA migrates further than regular DNA. I don't think it's less weight? but rather occupying a smaller space/volume?

@theonlytycrane, your thoughts?
 
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laczlacylaci

laczlacylaci

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This may be going off topic...

Well
|Nicked DNA (oc)
|Linear DNA (oc)
|Supercoiled DNA
|Closed-Circular DNA (ccc)

The reason why super coiled DNA & ccc migrates further is because it has less friction with the agarose gel, correct?
 

theonlytycrane

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@laczlacylaci Running a native gel gives a first indication of what the protein structure might be like in relation to something else. Maybe you run a native gel on two proteins and observe that one migrates further than the other even though we suspect they are of similar weight. That tells us that one of the proteins is more "compact" than the other because it's able to migrate a bit further on the gel. It's not much info, but it's a start.

If you run the same protein under non-reducing conditions, all of a sudden it might migrate less (this would make it seem heavier) or migrate more (this would make it seem lighter) because we've disrupted some of it's structure.

Finally, running a reducing gel gives us an idea about whether the protein is composed of multiple subunits held together by disulfide bonds. Using all of this information together gives us a more complete picture of the protein structure. Each individual piece of information alone isn't as useful.
 
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aldol16

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Hello, do you mind explaining this more? I don't quite understand. Why would it be either lighter or heavier?
Imagine this. Take a sheet of 8x11 paper and a rock of the same mass and drop it. Which one will hit the ground first and why (on Earth)? The rock will always hit the ground first because it presents less surface area to drag forces. Same with a folded vs. unfolded protein migrating through a gel (there are nuances depending on which type of tertiary structure it has). If the protein is spread out like a sheet of paper (predominantly beta-sheeted), it will appear to migrate slower due to drag forces. If it is clustered together like a rock (globular proteins), it will appear to migrate relatively faster than the alternative because of the minimal drag forces.

Also for that bottom gel titled non reducing and reducing, I notice that some of the lanes in non reducing gel is lower than the ones in reducing. Didn't you say that bands in reducing gels should be lower in weight?
Now apply the logic above. Say you have a disulfide that holds two strands of a protein together like a hairpin. Now if you reduce that disulfide, the protein migrates as one long chain. If you don't reduce the disulfide, the protein moves like a compact hairpin. It could be that the hairpin would migrate farther than the long strand, depending on various other factors. That's why you can't necessarily tell how reducing the protein will affect its migration.

The exception is when the disulfides are known to hold together subunits of the protein. If you reduce them, then instead of one 30 kDa unit, you get 20 kDa and 10 kDa subunits. These will appear to migrate faster in a reducing SDS-PAGE gel because they are lighter.
 
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