Electrophoresis

Question 1

What is electrophoresis? What is the equation that relates to electrophoresis?

Answer 1

the process by which charged proteins are separated in an electric field because of their differential mobilities

V = qE/f

V is movility

Charge of the molecule ( q )

Voltage gradient of the electric field ( E )

Frictional resistance of the supporting medium ( f )

Question 2

At the isoelectric point of a protein, what is the charge

Answer 2

neutral

Question 3

Why is a support medium needed? (2 things)

Answer 3

to prevent the loss of resolution and to
retain the separation of the proteins
after the completion of the experiment.

Question 4

Why is polymerized acrylamide used? (5 things)

Answer 4

Properties:

strong, hydrophilic, uncharged, stable, and adjustable porosity (that is controlled varying the percentage of acrylamide)

Question 5

Why is Sodium dodecyl sulfate (SDS) used? (what does it do?)

Answer 5

The caveat to separating proteins is that charge and size are parameters that affect migration and they both differ per protein (multiple independent variables)

So, to only separate based on size SDS is used

SDS is a detergent and denatures proteins. SDS has a net negative charge, and makes the proteins have a similar mass to charge ratio. This means that only size is variable now.

Question 6

What is the use for B-mecaptoethanol?

Answer 6

Breaks disulfide bridges

Question 7

What are the components of an SDS polyacrylamide gel (two gels, buffer and dye)

Answer 7

Stacking Gel: pH 6.8. Higher
porosity. Concentrate proteins
into a narrow zone. Glycine from
the buffer is neutral, faster mobility

▪Resolving or Separating Gel:
pH 8.8. Separation of proteins.
Glycine is anionic. Slower mobility

▪ Running Buffer: Tris-Cl pH 8.3
and glycine

▪Tracking Dye: BPB, high mobility, faster than proteins, determine end of the electrophoresis

Question 8

Applications for SDS page

Answer 8

▪Estimation of protein purity and quantitation. To track the
course of a protein purification, it is a good method to
visualize contaminants
▪ Determination of the molecular mass of proteins
▪ Western Blotting to detect and quantify proteins that react
with a specific antibody in particular physiological states or to
determine if a given protein is present.
▪ Purification of denatured proteins

Question 9

How to calculate Rf

Answer 9

Rf= Distance band travelled/distance dye traveled