5. Compare and contrast the sample buffers for DNA and protein electrophoresis.

6. Compare and contrast the running buffers for DNA and protein electrophoresis.

in protein electrophoresis

1.  Why do you
need to
wash the gel
before
staining it?
Why use
warm
water?
SDS running buffer

Tris-glycine Tris-glycine SDS: Tris base (25 mM), glycine (192 mM), SDS (0.1%), pH 8.3

Bis-Tris MES SDS: MES (50 mM), Tris base (50 mM), SDS (0.1%), EDTA (1 mM), pH 7.3
MOPS SDS: MOPS (50 mM), Tris base (50 mM), SDS (0.1%), EDTA (1 mM), pH 7.7

Tris-acetate Tris-acetate SDS: Tris base (50 mM), Tricine (50 mM), SDS (0.1%), pH 8.24

Tris-tricine

n SDS-PAGE, proteins are separated purely on the basis of molecular mass. All the other variables
are controlled. To understand how this works, you should understand what SDS-PAGE stands for.

SDS stands for Sodium Dodecyl Sulfate – but don't worry about remembering that; just call it
SDS. SDS is a detergent, and it is included in the buffers used in SDS-PAGE. This accomplishes
several critical things for electrophoresis:

 SDS helps proteins dissolve so you can run them on the gel (not all proteins are soluble in
plain water).
 SDS helps to denature, or unfold, proteins. This means that the relatively weak hydrogen
bonds, hydrophobic interactions, and ionic bonds that maintain the proteins' tertiary shape will
be undone, but the polypeptides' primary structure will not be altered. (Don't worry if you don't
yet understand primary and tertiary structure; these concepts will be covered in the first lecture
 unit.) The end result of using SDS is that proteins' shapes will not influence their rate of
migration in the gel.
 SDS sticks to proteins and makes them negatively charged. Since SDS sticks all over the
protein, each protein ends up with the same density of charge.
 In order to make this work, you'll have to pre-treat your protein samples with SDS and
include it in your gel. [For today's lab, you may be using Lithium Dodecyl Sulfate instead of
SDS; it's still called SDS-PAGE.]