Protein Electrophoresis

Question 1

Describe how proteins can have a charge.

Answer 1

Proteins can have either a net positive or negative charge. the charge depends on the pH of the buffer.

• in acidic solution, it has a net positive charge.
• in basic solution, it has a net negative charge

The backbone of the protein is uncharged. It is the R group that determines whether an amino acid is neutral, acidic or basic.

Question 2

What is electrophoresis?

Answer 2

It is the migration of charged molecules (macromolecules) in an electric field.

Question 3

List and describe the two types of electrophoretic gel.

Answer 3

AGAROSE:

• It’s a polysaccharide extract from seaweed.
• It’s prepared by dissolving powdered agarose in a buffer, heating then pouring into a casting tray. It undergoes polymerisation when cooled.
• Its pores are relatively larger, so it has a relatively low resolving power.
• It can be used to separate large proteins of over 200kDA
• It’s used at concentrations of 0.5-2%.

POLYACRYLAMIDE:

• It’s formed from the synthetic small molecule acrylamide.
• It polymerises into long chains in the presence of a catalyst and initiator (APS and TEMED).
• Polyacrylamide gels have smaller pores than agarose.
• The pore size is also determined by the polyacrylamide concentration.

Question 4

What is a buffer’s role in electrophoresis?

Answer 4

A buffer:

• supplies current carrying ions in electrophoretic cells
• maintains the desired pH
• provides a medium for heat dissipation

Question 5

What are the two classifications of buffers?

Answer 5

CONTINUOUS: uses the same buffer in the gel, sample and tank

DISCONTINUOUS: uses different buffers for stacking gel, resolving gel and the tank buffer

Question 6

Describe SDS-PAGE.

Answer 6

It stands for sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It is the most commonly used electrophoretic technique for separation. SDS has strong anionic detergent:

• to solubilise, dissociate and denature most proteins to single polypeptide chains
• to disrupt hydrogen bonds
• to block hydrophobic interactions

Here, the migration of the protein is not determined by intrinsic electrical charge, but by weight.

SDS-PAGE isn’t suitable for small polypeptides and peptides with a molecular weight of less than 10 kDa

Either a continuous or discontinuous buffer system can be used in SDS-PAGE.

Question 7

Describe native gel electrophoresis.

Answer 7

It’s used mainly in circumstances where native conformations are to be analysed. These native/non-denaturing gels run without SDS.
Here, proteins aren’t denatured. Separation is based on their:
- charge-to-size ratio
- conformation (shape)

The advantages to this are:

• the potential of separating proteins of identical molecular weight, not resolved with SDS-PAGE
• recovery of the protein in the native state
• you can study binding events (eg. protein-protein or protein-ligand)

Both agarose and polyacrylamide gels can be used in this electrophoresis. They are used for different sized molecules: agarose for ones >200 kDa, polyacrylamide for 5-2000 kDa.

Question 8

Give an example of the clinical applications of electrophoresis.

Answer 8

SERUM PROTEIN ELECTROPHORESIS (SPEP)
Blood is made up of blood cells and plasma. Plasma is made up of water, PROTEINS, salts, glucose, hormones, clotting factors, etc.

This electrophoresis measures specific proteins in the blood. SPEP uses an electrical field to separate proteins into groups of similar size, shape and charge. It helps to identify diseases.
Blood serum contains two major protein groups: Albumin and Globulins.

A densitometer is used to scan separated proteins in the gel. The pattern gives information about protein fractions.