Week 12 (biochemical Methods III)

Question 1

Describe the theory of electrophoresis

Answer 1

-A molecule with a net charge will move in an electric field
- The speed (velocity, v) at which the molecule moves depends upon;
-the strength of the electric field, E
-the net charge on the molecule, z
-the frictional coefficient, f (how much drag it experiences)
v = Ez/f

Question 2

Which gels is Electrophoresis is carried out in?

Answer 2

Polymer gels such as:
agarose
polyacrylamide

Question 3

What is the purpose of gels in electrophoresis?

Answer 3

These gels form a mesh like structure, which acts like a molecular sieve

Question 4

How do small/large molecules move through the mesh?

Answer 4

• small molecules can move through the mesh easily

- large molecules move slowly through the mesh

Question 5

How can electrophoresis be altered to give the best resolution for the molecules you are interested in?

Answer 5

-Can alter the concentration (%) of agarose/acrylamide in the gel
need to choose the concentration that gives the best resolution for the molecules you are interested in
-Can also make gradient gels – from 20% acrylamide at the bottom to 4% acrylamide at the top – can increase the resolution

Question 6

How far do large/ small molecules travel respectively?

Answer 6

• Smallest molecules travel furthest

- Large molecules move less

Question 7

what are the applications of electrophoresis?

Answer 7

Proteins
DNA
RNA

Question 8

What are the analytical applications of electrophoresis?

Answer 8

• Allows you to check the purity of samples
• Visualise the number of different molecules in a sample
• Estimate molecular mass

Question 9

What is the main advantageous property of electrophoresis?

Answer 9

Allows you to separate & visualise the components in your sample

Question 10

Explain Agarose gel electrophoresis based on the structure of DNA

Answer 10

• Phosphate groups in the backbone
• One charge per nucleotide, thus all fragments of DNA have the same ratio of charge to mass.
• Use agarose gels as these have larger pores (acrylamide pores are too small)

Question 11

Explain the process of agarose gel electrophoresis for DNA?

Answer 11

-Form wells using a comb before the agarose sets
-Mix DNA with loading dye and then add to wells
Loading dye contains:
-Glycerol – dense, causes sample to sink in the wells
-Bromophenol blue – a dye, allows you to track progress as the dye moves through the gel at the front

• In one well load a DNA ladder
• A series of DNA fragments of known length

Question 12

What is used to visualise the DNA?

Answer 12

• ethidium bromide
• A fluorescent dye that intercalates with the DNA
• Expose the gel to UV light

Question 13

SDS-PAGE for proteins

Answer 13

Proteins can have a wide range of different charges and shapes
Proteins can have a wide range of different shapes
Both of these can affect the velocity & migration of a protein in electrophoresis

Question 14

The shape of the protein can affect the velocity migration, how can this be overcome?

Answer 14

To overcome this we use SDS-Poly-acrylamide Gel Electrophoresis

Question 15

What does SDS stand for?

Answer 15

Sodium dodecyl sulphate

Question 16

What is SDS?

Answer 16

Is a detergent – denatures proteins
•loss of 2° & 3° structure
•only 1° structure remains

Question 17

Is SDS positively charged?

What does the charge mean for the proteins?

Answer 17

No
Is negatively charged
•large number of SDS molecules bound to each protein means all proteins have a large negative charge overall
•roughly constant mass to charge ratio

Question 18

Explain the process of SDS-PAGE for proteins

Answer 18

1. Mix protein sample with Laemmli sample buffer
2. glycerol – dense
   bromophenol blue – tracker dye
   SDS – denature protein & give it negative charge
   β-me/DTT – break disulphide bonds
3. Heat sample
   Ensures that full denaturation occurs
   Not good for membrane proteins because it makes them aggregate
4. Load on gel & apply voltage
   Include a lane of molecular weight standards

Question 19

What can be done to visualise the proteins?

Answer 19

use a stain such as Coomassie Blue

Initially whole gel goes blue

Destain (contains the same solvents as the stain but none of the dye) – blue disappears from the regions where there is no protein

The staining is fairly quantitative
More protein= more stain

Question 20

What is an alternative stain to Coomassie Blue?

Answer 20

silver stain – get black/brown staining of proteins
More sensitive than Coomassie- better for dilute samples
More expensive & time consuming than Coomassie

Question 21

What is the number of negative charges in DNA directly proportional to?

Answer 21

The size of DNA

Question 22

Where are the wells?

Answer 22

At the top

Biggest molecules at the top and the smallest at the bottom

Question 23

What can be done to break disulphide bonds (in the tertiary structure)?

Answer 23

Add a reducing agent

Question 24

Are SDS pages run vertically ?

Answer 24

Yes

Question 25

What would a homodimer produce?

Answer 25

A single band

Question 26

What would a heterodimer produce?

Answer 26

2 distance

Question 27

What is the molecular migration

Answer 27

Distance from log to band

Question 28

What do you do to find the molecular mass?

Answer 28

Add together the molecular weights of each band (kilo Daltons)