July 08, 2020, 05:54:45 AM
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Topic: Why is gel electrophoresis not linear? And why do small DNA/protein run faster?  (Read 4324 times)

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Offline poobear

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Hi,

1) We commonly do gel electrophoresis (agarose gels and SDS-PAGE). These can be run with different percentage of agarose and polyacrylamide. More agarose/polyacrylamide makes DNA/protein run slower. However, what I find interesting is that a gel with higher % run for a longer time will not look like a gel with lower % run for a shorter time. Different % are simply better for particular sizes of DNA/protein. I have briefly got it explained for me that this is because gel electrophoresis is logarithmic and not linear.
Is this correct?
And does anyone mind explaining it to me why the transfer in the gel is logarithmic? For me linearity is much more intuitive  :'( (or share link/paper that is easy to understand)

2) And why is larger DNA/protein running slower than smaller? Sure, smaller stuff more easily goes through the net of agarose/polyacrylamide. But smaller stuff also have less phosphates/SDS and so should have less attraction to the other pole. But as smaller DO indeed run faster, I guess the motility in the net matters more than the different attraction to the other pole. But this ratio (attraction to the other pole vs motility through the net) must change depending on the percent agarose/polyacrylamide right? If you go very low percentage, the more does the size of the DNA/protein become neglectable as it is so easy to go through the gel regardless of size. So is there then a theoretical percentage where small and big DNA/protein run at the same speed?

THANKS!

Offline Yggdrasil

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2) And why is larger DNA/protein running slower than smaller? Sure, smaller stuff more easily goes through the net of agarose/polyacrylamide. But smaller stuff also have less phosphates/SDS and so should have less attraction to the other pole. But as smaller DO indeed run faster, I guess the motility in the net matters more than the different attraction to the other pole. But this ratio (attraction to the other pole vs motility through the net) must change depending on the percent agarose/polyacrylamide right? If you go very low percentage, the more does the size of the DNA/protein become neglectable as it is so easy to go through the gel regardless of size. So is there then a theoretical percentage where small and big DNA/protein run at the same speed?

I'm not sure I know the answer to the first question, but I can answer your second question.  Larger DNA/protein molecules do experience a larger electrical force pulling them toward the anode.  This, as you correctly state, is due to the larger number of phosphates/SDS molecules on larger DNAs/proteins.

However, these larger DNAs/proteins also have more mass and therefore it takes more force to move them.  As an analogy, consider Galileo's famous experiment where he dropped a heavy ball and a light ball from the Leaning Tower of Pisa and demonstrated that they both hit the ground at the same time.  Even though the heavy ball experiences a greater gravitational pull from the Earth, it has more mass so the force imparts less acceleration on the ball.  Because the gravitational forces are proportional to the masses of the balls, the light and heavy balls fall at the same rate.

Similarly, because the amount of charge on the DNAs/proteins is proportional to their mass, molecules of any size would be expected to migrate through the gel at the same speed if there were no resistance from the gel.

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