Protein Electrophoresis

Question 1

What is SDS-PAGE used for?

Answer 1

Monitoring the progress of a purification procedure. To check the purity of the final product. To give a rough idea of the molecular weight of a protein (using molecular weight markers).

Question 2

What is the SDS used for in SDS-PAGE?

Answer 2

To denature proteins, forming nascent proteins- by disrupting hydrogen bonds and hydrophobic interactions.

Question 3

What are β-mercaptoethanol and DTT? Why would they be used in SDS-PAGE?

Answer 3

These chemicals are reducing agents which are used in SDS-PAGE to break any disulfide bonds in the proteins.

Question 4

What could multiple bands on an SDS-PAGE gel suggest?

Answer 4

Could be due to contamination or multiple subunits.

Question 5

Name two stains that could be used to visualise proteins in electrophoresis.

Answer 5

Coomassie blue (used for 1mg) and silver stain for low protein concentrations (1ng).

Question 6

How does Coomassie blue stain proteins?

Answer 6

Binds to lysine residues in the protein and gives a blue band in the gel.

Question 7

How does silver stain proteins?

Answer 7

Silver ions bind to the negative R groups in Asp and Glu residues, the sulfhydryl group in Cys and the imidazole ring in His.

Question 8

Explain the method of western blot and why it is used.

Answer 8

Used to detect proteins that have been separated by SDS-PAGE. Transfer from gel to nitrocellulose. Probe with an antibody specific to the protein of interest. Probe with a second antibody which binds to the first antibody and is linked to an enzyme. Add an enzyme-substrate chemiluminescent signal or a fluorescent signal.

Question 9

Why is a polymeric medium used (rather than free flow in solution)?

Answer 9

Convection would broaden the protein bands and limit resolution. The polymeric medium restricts diffusion as molecules diffuse more slowly when in a gel.

Question 10

What factors is the rate of migration proportional to?

Answer 10

Field strength, ionic strength, net charge, temperature, molecular size and shape and viscosity.

Question 10

Describe the polymer phase used in protein electrophoresis.

Answer 10

Usually acrylamide cross linked with methylene bis-acrylamide via radical induced polymerisation. Pore size is determined by the acrylamide concentration and the acrylamide:bis-acrylamide ratio. At 5% acrylamide there is no Mw sieving.

Question 11

Name the types of protein electrophoresis.

Answer 11

Denaturing, native, isoelectric focussing and 2D electrophoresis.

Question 11

What is migration dependent on in denaturing gels?

Answer 11

Only on size.

Question 12

How are proteins denatured in SDS-PAGE?

Answer 12

Heating to 100 degrees with beta-mercaptoethanol and SDS. SDS is bound on a constant weight basis - 1.4g SDS/g of peptide.

Question 13

Why do proteins only migrate by size in SDS-PAGE?

Answer 13

Charge on SDS overrides protein intrinsic charge, meaning that all proteins have the same charge and only migrate according to size.

Question 14

How do discontinuous gels improve resolution?

Answer 14

By reducing diffusional broadening. Fixes resolution damage caused by pipetting errors/proteins not being at the bottom of the wells.

Question 15

Describe the stacking gel in a discontinuous gel.

Answer 15

5% acrylamide, no Mw sieving, rapid migration. Found at the top of the gel near the wells.

Question 16

Describe the running gel in discontinuous gels.

Answer 16

12% acrylamide, Mw sieving, slower migration, separation of proteins.

Question 17

What happens to proteins during discontinuous gel electrophoresis?

Answer 17

They are concentrated at the stacking:running gel boundary before migrating according to size in the running gel.

Question 18

What does migration depend on in native electrophoresis?

Answer 18

Size and charge.

Question 19

What is native electrophoresis often used for?

Answer 19

Characterising complexes- determining Mw, stoichiometry and if proteins are monomeric.

Question 20

What conditions are used for native electrophoresis?

Answer 20

Ran at a high pH. The closer the pH is to the pI, the slower the migration and better the separation.

Question 21

What is the advantage of using charged dyes in native electrophoresis?

Answer 21

Allows more flexibility in pH.

Question 22

What is isoelectric focussing?

Answer 22

A high resolution electrophoresis method. Proteins migrate only according to pI.

Question 23

Explain the isoelectric focussing technique.

Answer 23

An ampholyte solution is incorporated into a gel. A stable pH gradient is established, using an electric field. The protein solution is added to the centre of the gel and the electric field is reapplied. After staining, proteins are shown to be distributed along the pH gradient according to their isoelectric points.

Question 24

What is protein electrophoresis used for?

Answer 24

As an analytical tool to test purity.

Question 25

What is a limitation of isoelectric focussing?

Answer 25

It is difficult to separate proteins with similar isoelectric points.

Question 26

What is 2D gel electrophoresis used for?

Answer 26

To separate proteins by both charge and mass, with high resolving power.

Question 27

Explain the 2D gel electrophoresis method.

Answer 27

An isoelectric focussing gel placed on an Polyacrylamide gel for SDS-PAGE. Isoelectric gel usually non-denaturing, only SDS-PAGE gel is denaturing. Splitting first by isoelectric point and then by mass.

Question 28

What is the relationship between mobility and charge/mass in 2D gel electrophoresis?

Answer 28

Mobility is directly proportional to charge. Mobility is inversely proportional to mass.

Question 29

What are the advantages of capillary electrophoresis?

Answer 29

High separation efficiency, small sample volumes can be used, can be automated and results can be quantified. Can be easily coupled to mass spectrometry.

Question 30

Give disadvantages of capillary electrophoresis.

Answer 30

Not suitable for low protein concentrations or large volumes. Hydrophobic molecules can be difficult to run effectively.

Question 31

What is meant by electrophoresis?

Answer 31

Differential movement or migration of ions by attraction or repulsion in an electric field.

Question 32

What is meant by electroosmosis?

Answer 32

Excess cations in the diffuse stern double layer flow towards the cathode, exceeding the flow towards the anode. Net flow occurs as solvated cations drag along the rest of the solution.

Question 33

Which effect is dominant in capillary electrophoresis?

Answer 33

The electroosmosis effect is dominant and overcomes the electrophoresis effect on negative ions going the other way- net flow towards the cathode.

Question 34

What is capillary IEF?

Answer 34

Separation in a pH gradient, proteins migrate to pI and then mobilised through the detector using high pressure or salt addition.

Question 35

Why must the electroosmotic force be suppressed in capillary IEF?

Answer 35

Want proteins to go in their native direction, and have no net flow towards the cathode unless prompted for detection.

Question 36

How is the electroosmotic force suppressed in capillary IEF?

Answer 36

The capillary is coated in Teflon.

Question 37

What gels are used for capillary electrophoresis?

Answer 37

Free solution gels- CZE and CGE

Question 38

What is used to set up the pH gradient in isoelectric focussing?

Answer 38

Ampholytes which are polyaminopolycarboxylic acids- have a range of pKas and buffer at a range of pHs.