Protein Gel Electrophoresis

Question 1

What are some of proteins crucial roles in biological processes ?

Answer 1

• Transport and Storage
• Mechanical support
• DNA/RNA metabolism
• Enzyme catalysis
• Control of growth and differentiation
• Immune response

Question 2

What do proteins differ in ?

Answer 2

• Size
• Shape
• Charge

Question 3

What is Electrophoresis ?

Answer 3

This is the process by which large charged molecules travel through a matrix under the influence of a uniform electric field

Question 4

What does the matrix act as ?

Answer 4

It acts as molecular sieve to aid separation

Question 5

What are some examples of these matrix ?

Answer 5

Polyacrylamide gel, agarose gel, paper and cellulose

Polyacrylamide gel electrophoresis (PAGE)

Question 6

How is polyacrylamide gel formed ?

Answer 6

By polymerisation of acrylamide

Question 7

Explain PAGE ?

Answer 7

• Mesh-like fiber network in three dimension

- Creates an environment that allows different sized proteins move through the gel at different rates

Question 8

How does PAGE separate proteins ?

Answer 8

According to their molecular weight

Question 9

However, proteins have different ?

Answer 9

They are different shapes, and have different charges

Question 10

What does SDS-PAGE stand for ?

Answer 10

Sodium Dodecyl Sulphate Poly-Acrylamide Gel Electrophoresis

Question 11

What is the aim in an experiment involving SDS-PAGE ?

Answer 11

To separate proteins according to their size i.e. chain length

Question 12

What is the problem ?

Answer 12

Proteins have secondary, tertiary or quaternary structure → wide range of shapes and charges

Question 13

So what is the solution ?

Answer 13

Use SDS to convert all proteins to the same shape and mask net charges

Question 14

What does an Anionic detergent do ?

Answer 14

• Disrupts non-covalent interactions (hydrogen bonds, hydrophobic interactions) thus unfolding the protein
• Binds most proteins in a constant fashion (about 1.4 grams of SDS per gram of protein)

Question 15

So, what does this ultimately do ?

Answer 15

• Masks net charge of the protein by forming large anionic complexes
• Minimises differences based on secondary or tertiary structure

Question 16

Why are reducing agents added to do ?

Answer 16

• Break disulphide bonds

- Detach subunits to ensure a separation based only on molecular weight

Question 17

So what does this ultimately to do the proteins ?

Answer 17

• Proteins have an homogenous negative charge
• Move to cathode when electric field is applied
• Larger proteins are more retarded.

Question 18

Gel has two layers that differ in their acrylamide concentration and pH. Explain them ?

Answer 18

• Stacking gel:
  low concentration acrylamide (4%), low pH (6.8)
• Resolving gel:
  higher concentration of gel (10-20%) and high pH (8.8, same as tank)

Question 19

What needs to be done so all proteins all enter the separation gel at the same time ? resulting in ?

Answer 19

Concentrate proteins in a narrow band in the interface between gels
This will result in a greater resolution of bands

Question 20

One acrylamide concentration only separates a fixed range of protein sizes: hence ?

Answer 20

Use a gradient for a broader range of separation

Question 21

The proteins migrate through ?

Answer 21

An increasing percentage of acrylamide (e.g. 4-20%)

Question 22

Thus the gel becomes increasingly ?

Answer 22

Restrictive for the proteins to migrate

Question 23

Most commonly used in ?

Answer 23

Lab, commercial pre-cast gels

Question 24

Small proteins run too fast in ?

Answer 24

Low percentage gels

Question 25

Gradient gels can separate?

Answer 25

A broader range of protein sizes

Question 26

Once the gel is run we need to visualise the proteins:

Answer 26

- Fix (immobilise) the proteins with a weak acid: proteins become insoluble and are thus prevented from diffusing during staining - Stain with Coomassie blue (or similar) dye to make protein bands visible - Then destain to remove excess dye

Question 27

Why use Non-denaturing gel electrophoresis (native-PAGE) ?

Answer 27

Separation according to: - Size - Net charge - Shape of native structure

Question 28

Native vs Denaturing gel run ?

Answer 28

In Native gel - All four levels analysed as secondary, tertiary and quaternary structure maintained However, denaturing gel (e.g. reducing SDS-PAGE) - Primary structure analysis only happens

Question 29

What is Western blotting used for ?

Answer 29

Technique for identifying a particular protein using antibodies after electrophoretic separation in a gel and transfer to a membrane.

Question 30

Explain the three steps ?

Answer 30

1. Separate proteins using SDS-PAGE 2. Transfer into nitrocellulose membrane 3. Stain and visualisation using antibody against protein of interest

Question 31

Why and how is two-dimensional gel electrophoresis used ?

Answer 31

1. Thousands of proteins in cells: too many to separate by SDS-PAGE gel alone 2. Use two dimensions, to separate the proteins on the basis of: - isoelectric point in the first dimension: isoelectric focusing - molecular weight in SDS-PAGE in the second dimension

Question 32

Low pH ?

Answer 32

Acidic groups neutralised

Question 33

High pH ?

Answer 33

Basic groups neutralised

Question 34

Change the pH, and what changes ?

Answer 34

The net charge changes

Question 35

What is the isoelectric point (pl) ?

Answer 35

pH at which the protein is neutral

Question 36

When is this different and what does this depend on?

Answer 36

- Different for different proteins | - Depends on amino acid composition

Question 37

At all pH values other than their pI, proteins will be?

Answer 37

Charged - at low pH, the protein is positively charged - at high pH, the protein is negatively charged

Question 38

Proteins accumulate at a pH region that corresponds to ?

Answer 38

Their pI, where they have no net charge and migration ceases.

Question 39

Result: proteins focused into ?

Answer 39

Sharp stationary bands with each protein positioned at its pI.

Question 40

At the isoelectric point the protein has no ?

Answer 40

No net charge and therefore no longer migrates in the electric field; for the protein shown the isoelectric is 6.5

Question 41

Explain Protein identification ?

Answer 41

- (Western blotting or) - Cut protein band out of gel - Digest with protease to make peptides - Identify by mass spectrometry

Question 42

What is Relative mobility (Rf ) ?

Answer 42

This is the distance migrated by a band divided by the distance migrated by the dye front (i.e. a small negatively-charged molecule).

Question 43

How can we measure Relative Mobility of Protein Bands ?

Answer 43

- Related to the protein’s molecular mass - Run a set of protein standards of known molecular mass - Draw a standard curve of log (mass) versus relative mobility - Estimate apparent mass for unknown proteins