L13: Electrophoresis

Question 1

What is electrophoresis?

Answer 1

Migration of ions in an electric field, widely used for separation of biological macromolecules (proteins)

Question 2

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What is the electric force of an ion (q) expressed by?

Answer 2

F(electric) = qE

Question 3

What is the electrophoretic migration of an ion through the solution expressed by?

Answer 3

F(friction) = vf

Question 4

Does the forces on the ion balance in a constant electric field?

Answer 4

Yes
qE = vf

Question 5

How is electrophoretic mobility (μ) expressed?

Answer 5

μ = v/E = q/f

Question 6

Most common gels that are used and what they are used for?

Answer 6

Polyacrylamide for proteins & agarose for nucleic acids

Question 7

Mesh size uses

Answer 7

Large -> larger pores -> better resolution for larger molecules

Smaller -> smaller pores -> better resolution for smaller molecules

Question 8

What is gel electrophoresis used for?

Answer 8

Separating macromolecules based on size & charge

Question 9

What does SDS-PAGE stand for?

Answer 9

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis

Question 10

How does gel electrophoresis separate biological macromolecules?

Answer 10

Based on their molecular masses

Question 11

What is the function of beta-mercaptoethanol in SDS-PAGE?

Answer 11

To break disulfide bonds in proteins

Question 12

What is SDS?

Answer 12

Negatively charged detergent that binds to proteins, unfolds them & gives them a uniform charge-to-mass ratio

Question 13

What is the purpose of using Coomassie Brilliant Blue in protein visualization?

Answer 13

To stain proteins for visualization

Question 14

How do proteins migrate during SDS-PAGE electrophoresis?

Answer 14

Based on their molecular masses

Question 15

What role does sodium dodecyl sulfate (SDS) play in SDS-PAGE?

Answer 15

To denature proteins and add negative charge

Question 16

What factors influence protein electrophoresis?

Answer 16

Molecular mass, protein charge, and isoelectric point

Question 17

What methods can be used to visualize proteins after electrophoresis?

Answer 17

Silver Staining

Question 18

What is agarose gel electrophoresis used for?

Answer 18

DNA & RNA separation

Question 19

Gel staining of agarose gel electrophoresis

Answer 19

Ethidium bromide (fluorescent under UV light)

Question 20

What is the significance of the isoelectric point (pI) in protein separation?

Answer 20

It is the pH at which a protein has NO net charge and can be separated from other proteins

Question 21

WHat is separation based on in isoelectric focussing (IEF)?

Answer 21

Separated based on their isoelectric point (pl)

Question 22

How is the pH gradient in isoelectric focussing formed?

Answer 22

Mixture of polyampholytes (small multicharged polymers)

Question 23

What is the importance of mesh size in polyacrylamide gel electrophoresis?

Answer 23

To determine the size of molecules that can pass through the gel

Question 24

How does two-dimensional gel electrophoresis enhance protein separation?

Answer 24

By separating proteins based on charge, then size

1) isoelectric focussing
2) SDS-PAGE

Question 25

Advantage of 2D gel electrophoresis

Answer 25

Resolves proteins with identical molecular weight but different charges

Question 26

4 ways to visualise proteins

Answer 26

1) Coomassie brilliant blue (CBB) 2) Silver staining 3) Ponceau red 4) Immunoblotting (western blotting)

Question 27

What is the process of blocking in immunoblotting, and why is it important?

Answer 27

Saturating the membrane to prevent non-specific antibody binding

Question 28

Use of ponceau red

Answer 28

Validating transfer to nitrocellulose after blotting

Question 29

What does immunoblotting need?

Answer 29

Needs primary antibody (binds target protein) & secondary antibody

Question 30

What is the purpose of immunoblotting in protein detection?

Answer 30

To detect specific proteins

Question 31

Function of primary/secondary antibody binding in immunoblotting

Answer 31

Primary: recognises target protein Secondary: recognises primary antibody

Question 32

Steps in the detection of proteins by immunoblotting

Answer 32

1) Nitrocellulose strip with tagret protein attached 2) Primary Ab binds to protein of interest 3) Secondary Ab binds to primary Ab, containing the HRP ennzyme 4) HRP enzyme reacts with chemiluminescent substrate & creates a glow that shows where the protein is on the membrane

Question 33

What is the function of chemiluminescence substrate in the detection process?

Answer 33

To produce light for detection

Question 34

What is the significance of using nitrocellulose in immunoblotting?

Answer 34

To act as a solid support for proteins

Question 35

What role does the primary antibody play in the immunoblotting process?

Answer 35

To specifically bind to the target protein

Question 36

How does the secondary antibody contribute to the detection of proteins?

Answer 36

It provides an enzymatic tag for detection

Question 37

What type of enzyme is commonly linked to the secondary antibody in immunoblotting?

Answer 37

HRP enzyme

Question 38

What is the importance of blocking unoccupied binding sites on nitrocellulose?

Answer 38

To reduce non-specific antibody binding

Question 39

Why might multiple chromatographic columns be needed for protein purification?

Answer 39

To separate proteins based on different properties for higher purity

Question 40

What factors determine the choice of purification method for a specific protein?

Answer 40

The protein's properties and the contaminants present

Question 41

What is the relationship between molecular weight markers and protein separation?

Answer 41

Markers estimate the sizes of separated proteins

Question 42

What is the purpose of affinity chromatography in protein purification?

Answer 42

To isolate a protein based on binding affinity

Question 43

How can the specific activity of a protein be calculated during purification?

Answer 43

Total activity / Total protein