W10 Electrophoresis

Question 1

isoelectric point (pI)

Answer 1

protein has no net charge at this pH, i.e. neutral (zwitterion)

Question 2

Electrophoresis

Answer 2

migration of charged particles (macromolecules) in electric field;

migration based on size, shape or charge

current and resistance

useful for separating /purifying macromolecules

Question 3

Macromolecules

Answer 3

consist of many subunits

each has multiple ionisable charged groups

Question 4

Electrophoresis apparatus

Answer 4

Two types:

Horizontal - usually for agarose gel

Vertical - for polyacrylamide gel

Question 5

What do you need to ensure when choosing apparatus?

Answer 5

uniform electric field across gel

cooling to prevent thermal artefact

access to gel loading and monitoring

Question 6

Gel Electrophoresis

Answer 6

Gel usually cast in shape of thin slab with wells,

immersed within buffer:

that provides ions to carry current,

maintain relatively constant pH,

pH of solution and nature of R-groups have important effect on migration of proteins,

proteins separated within gel with series of pores

Question 7

Agarose

Answer 7

Electrophoretic gel

polysaccharide extract from seaweed.

prepared by dissolving powdered agarose in buffer.

heat and pour into casting tray
undergoes polymerisation when cooled

pores relatively larger, so has relatively low resolving power

can be used to separate large proteins >200kDa

used at concentrations of 0.5 to 2%

Question 8

Polyacrylamide

Answer 8

formed from synthetic small molecule acrylamide

polymerises into long chains in the presence of catalyst and initiator (APS & TEMED).

polyacrylamide gels have smaller pores than agarose.

pores size also determined by polyacrylamide concentration

Question 9

Vertical slab gel electrophoretic apparatus

Answer 9

Most popular technique for protein electrophoresis

Question 10

Buffer

Answer 10

supplies current carrying ions in electrophoretic cell

maintains desired pH

provides medium for heat dissipation

Question 11

Buffer systems classified as

Answer 11

Continuous

Discontinuous

Question 12

Continuous buffer system

Answer 12

Uses same buffer in gel, sample and tank

Question 13

Discontinuous buffer system

Answer 13

Non-restrictive large-pore gel

Resolving gel – greater resolution

Have different buffers for stacking gel, resolving gel and tank buffer

Question 14

Protein Electrophoresis

Answer 14

Migration of any protein in electric field depends pI and pH

pI is constant for any given protein

pH of solution determines the charge expressed by protein;

e.g. Hb with pI of 7.07 donates proton to buffer if placed in electrophoretic buffer of pH 8.6

Question 15

SDS-PAGE

Answer 15

most commonly used electrophoretic technique for separation

SDS-PAGE includes disulfide bond cleaving agents (e.g. β-mercaptoethanol).

migration of protein not determined by intrinsic electric charge but by weight

Question 16

SDS has strong anionic detergent

Answer 16

to solubilise, dissociate and denature most proteins to single polypeptide chains.

disrupts hydrogen bonds

blocks hydrophobic interactions

binds at ratio of 1.4g SDS per gram of protein

conferring net negative charge to polypeptide in proportion to length

Question 17

Choice of Gel concentration

Answer 17

Gel concentration determines effective separation range of SDS-PAGE

SDS-PAGE not suitable for small polypeptide and peptides, of MW <10kDa

Continuous or discontinuous buffer system can be used in protein gel electrophoresis

Question 18

Detection Methods

Answer 18

Protein staining (in situ)
Coomassie brilliant blue dyes used as 0.1% (w/v) in methanol, distilled water and acetic acid (9:9:2,v/v/v)

Question 19

Native (non-denaturing) Gel electrophoresis

Answer 19

Used mainly in circumstances where native conformations are to be analysed

Native or non-denaturing gels run without SDS

Proteins not denatured, separation based on their,
charge-to-size ratio
conformation (shape)

Charge changes with change in pH of buffer

Question 20

Native (non-denaturing) Gel electrophoresis - advantages

Answer 20

potential of separating proteins of identical molecular weight not resolved with SDS-PAGE,

recovery of protein in native state,

study binding events (protein-protein or protein-ligand)

Question 21

Types of native gels

Answer 21

Agarose

Polyacrylamide

Question 22

Agarose pore size

Answer 22

do not have uniform pore size, but optimal for proteins > 200kDa (or nucleic acid >400bp)

Question 23

Polyacrylamide pore size

Answer 23

Polyacrylamide gels have uniform pore size, dependent on acrylamide and bis-acrylamide concentrations.
used for proteins sizes 5 – 2000 kDa

Question 24

Serum Protein Electrophoresis (SPEP) background

Answer 24

Blood made up of blood cells and plasma

Plasma is made up of water, proteins, salts, glucose, hormones, clotting factors

Question 25

Serum Protein Electrophoresis (SPEP) test

Answer 25

This measures specific proteins in blood;

SPEP uses electrical field to separate proteins into groups of similar size, shape and charge;

It helps to identify diseases

Question 26

Blood serum contains 2 major protein groups

Answer 26

Albumin

Globulins

Question 27

Densitometer

Answer 27

Used to scan separated proteins in the gel. The pattern gives information about protein fractions

Question 28

Haemoglobin Electrophoresis

Answer 28

pH range 8-9 (slightly alkaline) is most commonly used buffer system;

majority of proteins will be negatively charged