Electrophoresis

Question 1

Define electrophoresis

Answer 1

Migration of charged particles in an electrical field

Question 2

Describe the structure of an amino acid

Answer 2

Alpha carbon
Acidic carboxyl (-COOH) group
Basic amino (-NH2) group
Functional R group specific to each amino acid.

Question 3

Amino acids can exist as L- or D- forms. Which form is most common in proteins?

Answer 3

L- form

Question 4

In relation to amino acids, what is a zwitterion?

Answer 4

At the typical intracellular pH, many amino acids behave as both acids and bases. A molecule in which both the amino- and carboxyl- end are both ionised, but the molecule as a whole is electrically neutral (isoelectric) is called a zwitterion.

Question 5

If a molecule is isoelectric, it will not migrate in an electric field. How is this overcome during protein electrophoresis?

Answer 5

Altering the pH of the solution changes the charge on the amino acid.

Question 6

What does making the solution acidic do to the amino acid?

Answer 6

The carboxyl group gains a proton, giving the amino acid a positive charge, such that it will migrate to the negative pole.

Question 7

What does making the solution basic do to the amino acid?

Answer 7

The amino group loses a proton, giving the amino acid a negative charge, such that it will migrate to the positive pole.

Question 8

Genetic variations in proteins can alter the migration of proteins during electrophoresis, allowing the detection of these variants. How does this happen?

Answer 8

By substitution of a charged amino acid with a neutral amino acid, or vice versa.

Question 9

T or F: within the alpha-1 region, alpha-1 antitrypsin migrates cathodally

Answer 9

True. Alpha-1 lipoprotein (HDL) migrates anodally.

Question 10

Alpha 1 region proteins

Answer 10

Alpha 1 antitrypsin
Alpha fetoprotein
Alpha 1 antichymotrypsin
Alpha 1 acid glycoprotein

Question 11

Alpha 2 region proteins

Answer 11

Haptoglobin
Alpha 2 macroglobulin
Ceruloplasmin
Haemolysis
Fibronectin
Pre-beta1 lipoprotein (VLDL)

Question 12

Beta region proteins

Answer 12

Transferrin
Beta1 lipoprotein (LDL)
Complement C3, C4

Question 13

Gamma region proteins

Answer 13

Immunoglobulins
CRP
Fibrinogen (heparinised sample)

Question 14

Sebia Capillarys - dilution and wavelength and buffer pH and temp

Answer 14

1 in 10
200nm
10
35 deg

Question 15

Do proteins migrate from cathode to anode or anode to cathode in the Sebia Capillarys system?

Answer 15

Anode to cathode

Question 16

Why does albumin run most anodally on gel electrophoresis?

Answer 16

Because of its low MW (70000Da)

Question 17

Isoelectric Point of albumin

Answer 17

4.7

Question 18

MW of alpha and beta globulins?

Answer 18

40000->1000000Da

Question 19

MW of gamma/immunoglobulins?

Answer 19

150000->1000000Da

Question 20

Causes of pseudoparaproteins

Answer 20

1. Fibrinogen - incomplete clotting/plasma sample causing band at beta-gamma interface
2. Hemoglobin - haemolysed sample
3. Genetic or post-translational variants of eg TF, Hp, C3
4. CRP - large increases cause band in beta or gamma
5. Lysozyme - in monocytic leukemia may produce a band in the post gamma region
6. MAb therapy eg daratumumab

Question 21

Small molecules that may cause absorbance peaks in capillary zone electrophoresis?

Answer 21

Metabolites
Radiocontrast dyes
Drugs