Electrophoresis

Question 1

What are proteins?

Answer 1

A class of diversified biomolecules , differ in chemical properties like size, shape, charge, solubility etc.

Question 2

What are the functions of proteins?

Answer 2

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

Question 3

What is an isoelectric point?

Answer 3

At a certain pH, the molecule an be electrically neutral, I.e. negative and positive charges are equal

At this pH, it is the isoelectric point

Question 4

Which amino acids have negatively charged residues at physiological pH?

Answer 4

Glutamic acid and aspartic acid

Question 5

What amino acids have polar positive residues?

Answer 5

A protein’s positive charge at physiological pH is due to lysine, arginine and to a lesser extent, histidine

Question 6

What is the charge of amino acids at acidic pH?

Answer 6

• At an acidic pH, glutamic acid and aspartic acid residues have little charge, while lysine and arginine both have positive charges
• As the pH of the protein solution is raised, glutamic and aspartic acid release a proton and become negatively charged.
• However, lysine and arginine residues becomes uncharged as the pH is raised to high values

Question 7

What does amino acid sequence and post translational processing determine?

Answer 7

Proteins exhibit many different three-dimensional shapes and complex folding patterns

Question 8

Why is the 3 dimensional configurations of proteins important?

Answer 8

It is important to its biological function,

• general shapes for proteins are spherical, elliptical or rod-like
• the molecular weight is a function of the number and type of amino acids in the polypeptide chain

Question 9

What are native proteins?

Answer 9

Proteins that are in their normal, biologically active forms are called native

Question 10

What factors affect the migration rates of electrophoresis?

Answer 10

The magnitude of charge, the size and shape of a native protein

Question 11

What is serum?

Answer 11

Serum consists of many different types of proteins. Gel electrophoresis of native serum proteins at alkaline pH results in several zones

Question 12

Where is albumin synthesized?

Answer 12

In the liver

Question 13

What is the purpose of electrophoresis of native proteins ?

Answer 13

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

Question 14

What is the serum concentration and half life of albumin?

Answer 14

Serum conc.- 3.5-5.5 g/dL and a half life of 20 days

Question 15

What is the most abundant serum protein ?

Answer 15

Albumin, it also has one of the fastest electrophoretic migration rates

Question 16

What are the functions of albumin?

Answer 16

Albumin binds and transports many small molecules, including fatty acids and bilirubin

-it is also involved with osmotic regulation

Question 17

Which serum proteins with the slowest migration rates?

Answer 17

These are gamma globulins (antibodies)

Question 18

What is electrophoresis?

Answer 18

A technique used to separate, identify or purify macromolecules (e.g. proteins and nucleic acids) based on differences in:

• size
• charge
• conformation
• movement of charged particles through a medium (paper, gel) under the influence of an electric field

Question 19

What is the function of the supporting matrix in electrophoresis?

Answer 19

The frictional force of the supporting matrix acts as a “molecular sieve” , separating the molecules

Question 20

What happens In electrophoresis?

Answer 20

• 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
• the electrical current from one electrode repels the molecules while the other electrode simultaneously attracts the molecules

Question 21

A supporting matrix can be used for the electrophoresis of macromolecules and could be made up of:

Answer 21

-Paper, i.e. cellulose acetate paper

• Gel
  
  • Agarose or
  • Polysaccharide
  -gels used In electrophoresis (e.g. agarose, polyacrylamide) consist of microscopic pores of a defined size range that acts as a molecular sieve

Question 22

What is agarose?

Answer 22

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

Question 23

How are agarose gels made?

Answer 23

• suspending dry agarose powder 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 24

Which molecules will migrate through the gel when it’s in an electric field?

Answer 24

Tonly molecules with a net charge

Question 25

How does pore size affect molecule migration?

Answer 25

Small molecules will pass through the pores more easily than larger ones

Question 26

Molecules having a greater charge…

Answer 26

Than others of the same shape and size will migrate faster

Question 27

Molecules of the same mass and charge can have different shapes. Which will migrate faster?

Answer 27

In this case, those with a more compact shape, like a sphere, will migrate through the gel more rapidly than those with an elongated shape, like a rod

Question 28

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

Answer 28

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

Question 29

How should agarose be poured? And gel prepared?

Answer 29

• allow to cool and solidify for about 20 minutes
• remove rubber dams and comb after 20 mins when gel has set

Gel prepared

-nick gel to identify anode and cathode after staining

Question 30

How are protein samples loaded?

Answer 30

• insert casting tray and gel into electrophoresis chamber (tank) and fill chamber with buffer -pH 7.8
• load 25 uL of each sample into 5he appropriate well
• Cover tank, and connect lead wires to the power supply
• (Attach label w/ group number and the time you turned the power on)

Allow electrophoresis to run 125 V for 45 minutes

Question 31

How are proteins stained for visualization?

Answer 31

• stain for 60 minutes: solution contains 30 ml of protein plus stain + 270 ml of de-staining solution
• De-stain overnight: solution contains methanol, water and acetic acid - 450: 350: 100(9:7:2)