SDS-PAGE

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) is a process used to compare the sizes of proteins.

How SDS-PAGE works

Utilizing physics

Polyacrylamide gel electrophoresis utilizes the forces of an electric field and that of friction so that smaller proteins can travel further than larger proteins.

Setup

Be sure to plug the

wires into the correct

terminals!

Polyacrylamide GelUnlike DNA gel electrophoresis, agarose gel cannot be used for protein gel electrophoresis. Instead, the medium that is used is polyacrylamide gel.

Like all gels, polyacrylamide is a cross-linked polymer, which creates small tunnels. This allows smaller proteins to travel faster through the gel than larger ones.

In addition, polyacrylamide is inert, which means it will not denature the proteins.

http://www.bio.davidson.edu/courses/genomics/method/sdspage/PAgel.GIF

3 Factors

There are 3 main factors differentiating proteins as they travel through the charged gel medium:

● Size● Charge● Shape

Since SDS-PAGE is a procedure used to separate proteins by only size, then the proteins must not have different charges or shapes. Those 2 factors are “fixed” by the usage of SDS.

SDS–Sodium Dodecyl Sulfate

SDS, or sodium dodecyl sulfate, is an important molecule that is present in both the buffers and in the polyacrylamide gel.

It separates into anions and cations, the anion being sulfate. The anions attach to the proteins evenly, giving all of them a net negative charge, even if the proteins were originally positively charged.

Since the anions attach evenly to the proteins, all of the proteins now have the same charge-to-mass ratio, so proteins are not differentiated by this anymore.

Anions are negatively charged;

Cations are positively charged

SDS–Sodium Dodecyl Sulfate, pt. 2

SDS also ensures proteins do not have their tertiary structure (3-dimensional shape) and instead only have their primary structure.

This means that the proteins are simply a linear chain of amino acids; all of the treated proteins now have the same shape.

The only thing differentiating the proteins treated with SDS is how many monomers (amino acids) they each have.

Forces

Since the SDS was applied to the proteins, all of the treated proteins now have the same linear shape and the same charge to mass ratio. The only difference between different proteins is how many monomers they are composed of.

Because all of the proteins are also negatively charged, all of the proteins will head toward the positively-charged end of the gel.

Of course, the proteins cannot just freely move over to that end. There is friction exerted on the proteins by the polyacrylamide gel. Since larger proteins have more friction exerted on them, they will be held back more than the smaller proteins.

Setup+Process

1231 AAs121 kDa

Daltons (Da) are units used to

describe atomic mass.

1 Dalton= 1/12 of the mass of Carbon-12

1051 AAs[sdf] kDa