Lecture 7 – Electrophoresis

Question 1

Basic structure of proteins (10)

Answer 1

• Building blocks of proteins are amino acids residues.
• Two ionisable groups; amino gp N-terminus and carboxyl gp C-terminus.
• Amino acids are joined by peptide bonds.
• Proteins can have either a net –ve or +ve charge.
• Charge depends on pH of buffer.
• Isoelectric point (pI) – Protein has no net charge at this pH, i.e. neutral (zwitterion).
• pH < pI (acidic solution), net positive charge.
• pH > pI (basic solution), net negative charge.
• Backbone of protein not charged.
• R-group determines whether amino acid is neutral, acidic or basic.

Question 2

Principle of electrophoresis (5)

Answer 2

• Electrophoresis - Migration of charged particles (macromolecules) in electric field.
o Migration based on size, shape or charge.
o Current and resistance.
• Useful for separating /purifying macromolecules.
• Macromolecules consist of many subunits.
o Each has multiple ionisable charged groups.

Question 3

Electrophoresis apparatus (2)
What do you need to ensure when choosing apparatus? (3)

Answer 3

• Two types:
• Horizontal - usually for agarose gel.
• Vertical - for polyacrylamide gel.
• What do you need to ensure when choosing apparatus?
• Uniform electric field across gel.
• Cooling to prevent thermal artefact.
• Access to gel loading and monitoring.

Question 4

Gel electrophoresis - Overview (6)

Answer 4

• Gel usually cast in shape of thin slab with wells.
• Immersed within buffer:
o That provides ions to carry current.
o Maintain relatively constant pH.
o pH of solution and nature of R-groups have important effect on migration of proteins.
• Proteins separated within gel with series of pores.

Question 5

Gel electrophoresis - Agarose gel (7)

Answer 5

 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 6

Gel electrophoresis - Polyacrylamide (6)

Answer 6

 Formed from synthetic small molecule acrylamide.
 Polymerises into long chains in the presence of catalyst and initiator (APS & TEMED - tetra-methyl-ethylene-diamine).
 Polyacrylamide gels have smaller pores than agarose.
 Pores size also determined by polyacrylamide concentration.
 Vertical slab gel electrophoretic apparatus.
 Most popular technique for protein electrophoresis.

Question 7

Buffers (3)

Answer 7

• Supplies current carrying ions in electrophoretic cell.
• Maintains desired pH.
• Provides medium for heat dissipation.

Question 8

Buffer systems classified as … (6)

Answer 8

• Continuous
o Uses same buffer in gel, sample and tank.
• Discontinuous
o Non-restrictive large-pore gel.
o Resolving gel – greater resolution.
o Have different buffers for stacking gel, resolving gel and tank buffer.

Question 9

Protein electrophoresis (3,1)

Answer 9

• 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 10

SDS-PAGE (Sodium Dodecyl Sulphate-Polyacrylamide gel electrophoresis) (9)

Answer 10

• SDS-PAGE: most commonly used electrophoretic technique for separation.
• SDS has strong anionic detergent:
o To solubilise, dissociate and denature most proteins to single polypeptide chains.
o Disrupts hydrogen bonds.
o Blocks hydrophobic interactions.
o Binds at ratio of 1.4g SDS per gram of protein.
o Conferring net negative charge to polypeptide in proportion to length.
• SDS-PAGE includes disulphide bond cleaving agents (e.g. β-mercaptoethanol).
• Migration of protein not determined by intrinsic electric charge but by weight.

Question 11

Movement of macromolecules (1)

Answer 11

• In an electric field, negatively charged molecules migrate towards positive pole (anode).

Question 12

Choice of gel concentration (3)

Answer 12

• 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 13

Detection methods (4)

Answer 13

• 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).
• Fluorescent staining.
• Silver staining.
• Radioactive methods (radiolabelling, autoradiography, fluorography).

Question 14

Native (non-denaturing) Gel electrophoresis - Overview (6)

Answer 14

• 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:
o Charge-to-size ratio.
o Conformation (shape).
• Charge changes with change in pH of buffer.

Question 15

Native (non-denaturing) Gel electrophoresis - Types of native gels (2)

Answer 15

 Agarose
Agarose do not have uniform pore size, but optimal for proteins > 200kDa (or nucleic acid >400bp).
 Polyacrylamide
Polyacrylamide gels have uniform pore size, dependent on acrylamide and bis-acrylamide concentrations. Used for proteins sizes 5 – 2000 kDa.

Question 16

Native (non-denaturing) Gel electrophoresis - Advantages (3)

Answer 16

o Potential of separating proteins of identical molecular weight not resolved with SDS-PAGE.
o Recovery of protein in native state.
o Study binding events (protein-protein or protein-ligand).

Question 17

Clinical Applications of Electrophoresis - Serum Protein Electrophoresis (SPEP) (8)

Answer 17

• Blood made up of blood cells and plasma.
• Plasma is made up of water, proteins, salts, glucose, hormones, clotting factors.
• 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.
• Blood serum contains 2 major protein groups:
o Albumin
o Globulins

Question 18

Clinical Applications of Electrophoresis - Haemoglobin Electrophoresis (1)

Answer 18

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