Gel Electrophoresis

[regeneration]

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Hi guys,

A bit confused: I understand that a smaller fragment (of DNA or protein w/ SDS) is supposed to run faster in a gel than a larger fragment; this is because the pores in the gel allow for smaller fragments to move more quickly.

However, why is it that larger fragments with more charge don't have a greater attraction to the positively charged anode? I was thinking that since the larger fragments have more charge, they should have more force pulling them.

Thanks.

 

[Captain Sisko]

Hi guys,

A bit confused: I understand that a smaller fragment (of DNA or protein w/ SDS) is supposed to run faster in a gel than a larger fragment; this is because the pores in the gel allow for smaller fragments to move more quickly.

However, why is it that larger fragments with more charge don't have a greater attraction to the positively charged anode? I was thinking that since the larger fragments have more charge, they should have more force pulling them.

Thanks.

Here's my understanding of it, but that doesn't mean it's 100% correct. Others should definitely weigh in since biochem methods aren't my strong suit.

The answer is, is that larger fragments with more charge will get attracted more strongly, however this increased force is much smaller than the viscous drag forces that are due to the gel itself. So the force due to an electric field is F = Eq, where q is the charge. But the force due to viscous drag is F = cd*A*(.5 density*velocity^2) where A is the cross sectional area of the item being analyzed, cd is the drag coefficient (dependent on shape of the molecule) and density is that of the gel. A goes up faster than charge, since for DNA you have a negative charge for every phosphate group, but A increases much faster since for every phosphate group you have a whole nucleoside to go with it.

For proteins and SDS, SDS is a soap that imparts a negative charge to the denatured protein so it will attract the anode. But the same logic above applies.

So what this all says is that the relationship between charge and distance run on a gel won't be direct since you've two different forces, one of which depends on the velocity, which in turn depends on the size. But what it boils down to is that size is the dominant factor, which is all what we need to know.

hope that helps and wasn't confusing, and is at least somewhat right.

 

[ChemTutor]

The key idea is that macromolecules during SDS-PAGE for proteins and Agarose GE for DNA have a uniform mass/charge ratio. I guess when the above mentioned formulas are worked out the result is that the migration distance is proportional to -log Mr.

 

[chem4ever]

Hi guys,

A bit confused: I understand that a smaller fragment (of DNA or protein w/ SDS) is supposed to run faster in a gel than a larger fragment; this is because the pores in the gel allow for smaller fragments to move more quickly.

However, why is it that larger fragments with more charge don't have a greater attraction to the positively charged anode? I was thinking that since the larger fragments have more charge, they should have more force pulling them.

Thanks.

I am actually super excited that you asked this question because I was asking myself the same thing yesterday and it was driving me crazy until I had that AHA moment!

SDS causes there to be a perfect (well almost perfect) mass to charge ratio. So let's say a 100 dalton protein has 1 coulomb of charge (very unrealistic numbers but just trying to get my point across) after SDS was added, then a 200 dalton protein in the same mixture will have 2 coulombs of charge.

Now, when added to the gel electropheresis, the 200 dalton protein will feel a greater force (like you said) because it has a greater charge. Now we have to relate the force to the protein's acceleration through the gel. We know that F=ma, so that means that F/m=a. Since for the 200 dalton protein, the F will be exactly double (the F for the 100 dalton protein), and the m is also exactly double, that means that the acceleration will be the exact same.

In other words, if the 200 dalton protein had an F of 10 N, then the 100 Dalton protein would have an F of 5 N (exactly half because the charge is exactly half). Then a = F/m = 10/200 = 1/20 m/s^2 for the 200 dalton protein and 5/100 = 1/20 m/s^2 for the 100 dalton protein. So they will both have the exact same acceleration.

Now that they are both accelerating the same, the only thing that is stopping them is the pores which catch on to the larger proteins more than they catch on to the smaller proteins. Thus, the larger proteins will not travel as far.

The whole point of SDS, is to take the charge out of the equation. By adding SDS, you make the size of the protein the only factor in how far the protein travels.

Let me know if you need better clarification.

 

[regeneration]

^Thanks! That makes sense for proteins coated with SDS. I had a feeling that might have been the case.

Captain, so you're saying for DNA, where the charge:mass ratios are different, the forces felt are different but that drag force is significantly greater; and this would cause larger DNA molecules (w/ bigger surfaces area) to go slower through the gel even though there's a larger force pulling on it?

 

[chem4ever]

^Thanks! That makes sense for proteins coated with SDS. I had a feeling that might have been the case.

Captain, so you're saying for DNA, where the charge:mass ratios are different, the forces felt are different but that drag force is significantly greater; and this would cause larger DNA molecules (w/ bigger surfaces area) to go slower through the gel even though there's a larger force pulling on it?

In DNA, the charge:mass ratio is not different. Each base has a single negative due to the phosphate group. So even without adding something like SDS, the DNA already has a perfect mass:charge ratio.

 

[regeneration]

In DNA, the charge:mass ratio is not different. Each base has a single negative due to the phosphate group. So even without adding something like SDS, the DNA already has a perfect mass:charge ratio.

Oh, yeah, of course. Thanks 👍