Electrophoresis

Question 1

Electrophoresis

Answer 1

method to separate molecules on the basis of their charge and size; Positively charged molecules will move towards the cathode while negatively charged molecules move towards the anode.

Question 2

What affects Migration Rate

Answer 2

Depends on the strength of the electric field; ionic strength, viscosity, and temperature of the medium in which the molecules are moving also affect the migration rate of the molecules

Question 3

Equation for Migration Rate

Answer 3

v’=charge/mass

Question 4

Paper electrophoresis

Answer 4

amino acid and nucleotides

Question 5

Polyacrylamide gel (PAG)

Answer 5

: proteins and nucleotides

Question 6

Agarose gel

Answer 6

: very large proteins, DNA

Question 7

Tracking Electrophoretic Movement

Answer 7

Proteins and DNA are negatively charged and will move to the anode. Bromophenol blue is added to the sample, it is a negatively charged dye and therefore migrates in the same direction as a protein or DNA and track the extent of the electrophoretic movement

Question 8

DNA staining

Answer 8

Proteins and DNA are colourless, so visualising agent (dye) that binds to their structure is added to the buffer or the gel; for proteins - coomassie blue (binds to proteins), for DNA - ethidium bromide (binds to DNA helix)

Question 9

Isoelectric Focusing

Answer 9

pH gradient formed during electrophoresis, so t a pH value, the protein reaches its isoelectric point (pI) and carries a zero net charge, the protein molecules stop moving and become focused in a narrow zone on the gel

Question 10

PAG

Answer 10

acrylamide monomers are polymerized into long chains that are covalently linked by N, N’ methylenebisacrylamide
PAG gels are very stable

Question 11

Agarose:

Answer 11

is a linear polymer extracted from seaweed.
polymer chains are held together by the formation of weak hydrogen and hydrophobic bonds

Question 12

SDS-PAG:

Answer 12

A gel electrophoresis method to separate proteins based on the their mass
In SDS-PAG, the disulfide bonds between the polypeptide chains are first reduced using 2-Mercaptoethanol
Sodium dodecylsulfate (SDS) is added. The anionic detergent denatures the structure of the protein by wrapping around the polypeptide backbone and breaking the hydrogen bonds to unfold the protein.
The SDS also creates a net negative charge on the polypeptide chains and they carry equal charge densities
Short polypeptide chains fit easily and move fast through the pores of the PAG gel, while larger ones move slowly