Electrophoresis Flashcards Preview

AP0511 Molecular Biology and Genetics > Electrophoresis > Flashcards

Flashcards in Electrophoresis Deck (40)
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1
Q

What is electrophoresis?

A

A separation technique based on the movement of charged molecules in an electric field.

2
Q

Different molecules move at different rates through the gel based on what?

A

Their net charge, size, shape and electric field strength.

3
Q

Name 3 applications of electrophoresis?

A

Separation of nucleic acids in research and genetic diagnostics
Separation of proteins in research and diagnostics
Separation of small charged molecules (amino acids, pharmaceuticals)

4
Q

What was the very first electophoresis technique in the 1930s?

A

Separating plasma proteins by moving boundary electrophoresis carried out in a U-shaped tube. But it had low resolution due to diffusion and convection currents.

5
Q

Explain the net charge in an elecrtophoresis set up and how this lets charged molecules move across the gel.

A
The anode (+ electrode) lets negatively charged molecules (anions) move towards it.
The cathode (- electrode) lets positively charged molecules (cations) move towards it.
Higher charged molecules move faster than smaller charged.
6
Q

How does the size and shape affect the movement of molecules through the gel?

A

Smaller molecules (in size and shape) move faster through the gel than bigger ones.
Linear DNA/circular DNA move at different speeds.
Globular/fibrous proteins move at different speeds.

7
Q

How does field strength (voltage) affect mobility of molecules?

A

Mobility increases with increasing field strength (until heating effects occur that reduce mobility).

8
Q

What does the equation for electrophoretic mobility mean? (EM= Eq/r)

A

E= electric field strength
q= net charge of molecules
r= radius of molecules (size+shape)
the mass:charge ratio affects the movement of molecules based on field strength.

9
Q

What reaction occurs during electrophoresis and how is it stopped?

A

Electrolysis occurs and is stopped by switching off the electric current before molecules reach the electrodes.

10
Q

Heating effects occur during electrophoresis. Why is this a problem?

A

Problems:
Causes convection currents which can lead to zone broadening by increased diffusion of sample and buffer.
Heat denatures samples like proteins and enzymes
Heat reduces buffer viscosity leading to reduced frictional resistance.

11
Q

How are heating effects in electrophoresis avoided?

A

Use a power pack that provides constant power

Use a cooling device like a water cooling system

12
Q

Why are supporting media used and give examples of supporting media?

A

To minimize diffusion and convection problems.

Paper, starch, agarose, cellulose acetate and polyacrylamide.

13
Q

Cellulose Acetate is a media type used. Describe some of its characteristics.

A

Less hydrophilic than cellulose so holds less water.
Allows reduced diffusion with increased resolution.
Uniform with large pores
No molecular sieving

14
Q

Agarose Gel is a media type used. Describe some of its characteristics.

A

Made from seaweed.
A linear polysaccaride.
Low concentration gives large pores
High concentration gives smaller pores

15
Q

Polyacrylamide Gel is a media type used. Describe some of its characteristics.

A

Made by cross-linking polymerised chains of acrylamide.

Pore size is determined by concentration of acrylamide.

16
Q

Which supporting media type is used for ROUTINELY separating DNA fragments?

A

Agarose Gel Electrophoresis.

17
Q

Which supporting media type is used to separate DNA at HIGHER RESOLUTION?

A

Polyacrylamide Gel Electrophoresis

18
Q

Why is DNA only separated based on length (size) and not charge?

A

All DNA has the same mass:charge ratio as the phosphates make all DNA negatively charged. So they are separated based on size alone.

19
Q

What is the routine for Agarose Gel Electrophoresis?

A

1) Agarose is boiled in electrophoresis buffer until dissolved.
2) Then it is poured into casting tray with sealed ends and a comb inserted
3) Let cool then take out comb to reveal wells
4) Fill tank with buffer solution
5) DNA samples mixed with loading buffer (contains dye) are loaded into separate wells
6) Turn on electric field at 60V and wait 90 mins
7) Read off results

20
Q

What is the routine for PAGE (polyacrylamide gel electrophoresis)? Used to separate very small DNA fragments.

A

1) Vertical glass plates separated by thin spacers with a comb in is set up.
2) Gel is poured in between the glass plates and let cool
3) Remove comb and load samples of DNA
4) Loading buffer contains dye bromophenol blue and DNA is stained after using ethidium bromide.

21
Q

How does Pulsed Field Gel Electrophoresis work? For separating very large DNA fragments.

A

Electric field is applied alternately at different angles for different times. Agarose gel is used. Allows whole chromosomes to be separated.

22
Q

What determines how proteins move through gel in electrophoresis?

A

Net charge of a protein is pH-dependant and the amount of positive and negative side chains on the amino acids.

23
Q

What are the advantages of PAGE when used for protein electrophoresis?

A

versatility in terms of pore size
chemically inert matrix
stable over a wide range of pH values, ionic strength of amino acids
is transparent (suitable for scanning)

24
Q

What is the most widely used stain in protein electrophoresis?

A

Coomassie blue R-250

25
Q

What stain is used for greater sensitivity of detecting proteins (and DNA and polysaccharides)?

A

Silver staining but is more expensive

26
Q

What is the difference between dissociating and non-dissociating PAGE for protein separation?

A

Dissociating PAGE- proteins are denatured and dissociated using detergent that breaks disulphide bonds.
Non-dissociating PAGE- proteins remain in their normal conformations

27
Q

What detergent is universally used in dissociating PAGE/ denaturing gel electrophoresis for protein analysis?

A

Sodium dodecyl sulphate (SDS)
It makes polypeptide charge proportional to its length so proteins are separated by length and not charge.
Create a net negative charge, dissociating all proteins into their individual polypeptide subunits.

28
Q

What are size standards/ markers?

A

polypeptides/DNA fragments of known lengths/molecular mass that are used as reference to analyse sample sizes. Can be analysed by plotting relative Mr against the mobility of proteins/DNA through gel.

29
Q

How do Gradient polyacrylamide gels work?

A

They have a gradient of pore sizes across the length of the gel so that a wider range of Mr proteins can be separated.
Goes from higher to lower pore sizes.
Allows for zone sharpening.

30
Q

How are gels analysed? (3 ways)

A

1) Instruments like laser densitometers
2) Gel scanning (measure absorbance of coomassie blue stain at 560nm)
3) Record gel using digital camers above white glass transilluminator

31
Q

Although not done anymore, how can gels be stored?

A

Preserved in acetic acid or dried using gel driers and stored at room temperature.

32
Q

How does Isoelectric Focusing (IEF) be used to separate out proteins differing by 0.01 isoelctric points?

A

IEF uses ampholytes (small molecular mass compounds) placed between anode and cathode. Then electric field applied and ampholytes migrate to their isoelectric points (pI). Forms a pH gradient.
Sample run through polyacrylamide gel and the narrow pH gradient allows sensitive seaparation of isoelectric proteins.

33
Q

What is the advanced electrophoretic technique of Two-Dimensional Electrophoresis?

A

2D electrophoresis is most commonly used to separate protein samples of 1000 proteins.
Uses IEF to separate out proteins by charge in first dimension.
Then uses SDS-PAGE to separate proteins by molecular mass in second dimension.

34
Q

What is used in the first dimension of 2D electrophoresis?

A

Polyacrylamide rod gels that are very thin glass tubes. After sample is resolved, the gel must be removed from the rod by cracking the glass, freezing it or squirting buffer through it.

35
Q

What is used in the second dimension of 2D Electrophoresis?

A

Polyacrylamide slab gel whose size is determined by the length of the glass rod used in first step. SDS-PAGE buffer used and is loaded between glass plates.

36
Q

What staining is used for 2D Electrophoresis?

A

coomassie blue or silver stain (for higher sensitivity)

37
Q

How are 2D Electrophoresis Gels analysed?

A

Very complex so are analysed using computer-aided gel scanners, which allows us to quantify individual proteins. Use a database and markers to identify the proteins.

38
Q

What is Capillary Electrophoresis?

A

Combination of high resolving power of electrophoresis with speed and versatility of HPLC (high-performance liquid chromatography).

39
Q

What are the advantages of Capillary Electrophoresis?

A

Removes issues like poor resolution, diffusion, heat due to electric current.
Very small samples can be used.
Wide range of biomolecules can be analysed

40
Q

What is the principle behind Capillary Electrophoresis?

A

Uses Electro-osmotic flow.