Electrophoresis

Question 1

What is electrophoresis?

Answer 1

Technique used to separate, identify or purify macromolecules (e.g. proteins and nuclei acids) based on differences in

Size

Charge

Conformation

Question 2

What does electrophoresis cause?

Answer 2

Movement of charged particles through medium (paper, gel) under the influence of an electric field

Question 3

Explain the process of electrophoresis

Answer 3

• When charged molecules are placed in an electric field, they migrate(through a stationary solid medium) towards either the positive (anode) or negative(cathode) electrode
• current from one electrode repels molecules away while the other attracts the molecule
• Frictional force of the supporting matrix acts as a “molecular sieve”, separating the molecules

Question 4

What do electrophoresis gel consist of?

Answer 4

Microscopic pores of a defined size range that act as a molecular sieve

Question 5

What are options for electrophoresis gels?

Answer 5

Agarose

Polyacrylamide

Question 6

What are options for supporting matrix?

Answer 6

Paper( cellulose acetate paper)

Gel

• agarose
• polyacrylamide

Question 7

What is agarose?

Answer 7

A polysaccharide extracted from seaweed and used at concentrations of 0.5 to 3%

Question 8

How are agarose gels made?

Answer 8

• suspending dry agarose power in aqueous buffer, then boiling the mixture until a clear solution is formed.
• Clear solution(at 55 degrees Celsius) is poured into a casting tray and allowed to cool to room temperature and solidify into a gel

Question 9

In electrophoresis, only molecules with…

Answer 9

A net charge will migrate through the gel when it is in an electric field

Question 10

Which travels through the pores faster, small or large molecules?

Answer 10

Small molecules pass through pores more easily

Question 11

Two molecules with the differing charge but same size and shape are placed in electrophoresis, which moves faster?

Answer 11

The one with more charge

Question 12

Molecules with same mass and charge it differ in shape are placed in electrophoresis, which moves faster?

Answer 12

The one with a more compact shape, such as a sphere, migrate through the gel more rapidly than those with an elongated shape, such as a rod

Question 13

What does the rate of migration through the electric field depend on?

Answer 13

• strength of the field(voltage)
• size and shape of the molecules
• relative hydrophobicity of the samples
• concentration of gel
• ionic strength and temperature of the buffer molecules are in

Question 14

What are the functions of proteins?

Answer 14

• Enzyme catalysis
• metabolic regulation
• binding transport of small molecules
• gene regulation
• immunological defense

Cell structure

Question 15

What enable proteins to carry out their biological functions?

Answer 15

Charge, shape, size and solubility

Question 16

What are the functions of proteins?

Answer 16

Enzyme catalysis

Metabolic regulation

Binding and transport of small molecules

Gene regulation

Immunological defense

Cell structure

Question 17

How does electrophoretic separation of proteins work?

Answer 17

A protein at a negative or positive charge will migrate towards the electrode of opposite charge

Question 18

What amino acids are due to a polar, negative charge at physiological pH ?

Answer 18

Glutamic acid and aspartic acid

Question 19

Which proteins give a polar, positive charge at physiological pH

Answer 19

Lysine, arginine and to a lesser extent histidine

Question 20

Contrast acidic and basic proteins in an acidic pH

Answer 20

Glutamic acid and aspartic acid have little charge

Lysine and argunine have positive charges

Question 21

Compare acidic and basic proteins when the pH becomes more basic/raises

Answer 21

• Glutamic and aspartic acid become released a proton and become negatively charged
• Lysine and arginine residues become uncharged pH is raised to high values

Question 22

Proteins exhibit many 3D shapes and complex folding patterns, what is this determined by?

Answer 22

Their amino acid sequence and post transitional processing such as adding prosthetic groups

Question 23

The precise three dimensional configuration of a protein is critical to….

Answer 23

It’s biological function

Question 24

What are the general shapes of proteins?

Answer 24

Spherical, elliptical or rod-like

Question 25

The number and nature of polypeptides in a protein has large effects on…

Answer 25

It’s mass, size and shape

Question 26

What is the native form of a protein?

Answer 26

The normal, biological active form of a protein

Question 27

What effect migration rates of a protein?

Answer 27

The amount and sign of a charge, the size and shape of a native protein, all effect it’s electrophoretic migration rates

Question 28

What would be the purpose of electrophoresis of native proteins?

Answer 28

Useful clinical and immunological analysis of complex biological samples such as serum

Question 29

Discus albumin as a serum in electrophoresis

Answer 29

By far the most abundant serum protein and has one of the fastest electrophoretic migration rates

Question 30

Discuss serum as a a substance tested in gel electrophoresis

Answer 30

Native serum in electrophoresis at alkaline pH results in several zones

Question 31

What are the functions of albumin

Answer 31

Binds and transports many small molecules, including fatty acids and bilirubin

Involved in ismoregulation

Question 32

Which serum proteins has the slowest migration rates?

Answer 32

Gamma globulins(antibodies)

Question 33

What proteins can be observed between albumin and gamma globulins?

Answer 33

Transferrin(ion transfer)

Ceruloplasmin(copper transport)

Macroglobulin(protease inhibitor)

Haptoglobin (binding and conservation of hemoglobin)

Question 34

How can electrophoretic patterns of human serum protein said in diagnosis of diseases?

Answer 34

• Cirrhosis of liver causes decrease in albumin
• Multiple myeloma(cancer of the immune system) and chronic rheumatoid arthritis causes abnormal increases in the gamma globulins

Question 35

Recall the steps of gel electrophoresis

Answer 35

Remove rubber dams and comb after 20 minutes when gel has set

Insert casting tray and gel into electrophoresis chamber(tank) and fill chamber with buffer-7.8

Load 30ul of each sample into the appropriate well

Cover tank and activate power source(run at 125V for 45 mins)

Stain proteins