Brainscape's Knowledge GenomeTM

Browse over 1 million classes created by top students, professors, publishers, and experts.


See full index

MADD > Electrophoresis > Flashcards

Electrophoresis Flashcards

1
Q

What happens when an electric field is applied?

A

Charged species begin to move

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
2
Q

Where do cations move towards?

A

Cathode

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
3
Q

What does the rate of migration depend on? (x3)

A
  1. Net charge of molecule
  2. Size and shape of molecule
  3. External environment (buffer pH, electric field and temperature)
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
4
Q

What is electrophoretic mobility defined as?

A

The average velocity with which an ion moves in an applied electric field (=V/E)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
5
Q

What does the electric force (Fe) depend on?

A

The charge and the (applied voltage/distance between electrodes)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
6
Q

What is movement opposed by, and what is this proportional to?

A

Frictional drag - exerted on the charged particles by the medium and is proportional to the velocity of the particle

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
7
Q

What happens once electrophoresis has started?

A

There is rapid acceleration of the ions and equilibrium is reached within milliseconds

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
8
Q

What happens to the resultant force at equilibrium?

A

It is 0 and the molecules move at a constant velocity

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
9
Q

What can mobility be interpreted as?

A

Proportional to charge-to-size ratio

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
10
Q

Describe the setup in capillary electrophoresis

A

A fused-silica capillary with an optical viewing window, a high voltage power supply, two electrodes, two buffer reservoirs and a detector

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
11
Q

What molecules in particular can be separated in CE?

A

Polar molecules

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
12
Q

What is the capillary filled with?

A

Buffer

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
13
Q

How is the sample injected in CE?

A

Electrokinesis or pressure

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
14
Q

What happens after electric field is applied in CE?

A

An electric double layer is produced at the capillary surface due to the attraction of positively charged ions in the buffer to the ionised silanol groups at the capillary wall

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
15
Q

In the presence of an electric field, what happens to the cations in the diffuse (outer) portion

A

Move to the cathode and drag the solvent with them, producing an electroosmotic flow (EOF)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
16
Q

What is formed due to different electrophoretic mobilities of ionic species?

A

Zones of analytes that migrate toward the outer side of the capillary at different rates

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
17
Q

What does rate of migration depend on in CE?

A

Mobility is proportional to q/f, which is approximately equal to the m/z ratio

18
Q

What does electrophoresis refer to in GE?

A

The electromotive force (EMF) that is used to move molecules through a gel matrix

19
Q

What is the gel?

A

A matrix used to separate proteins

20
Q

What is the gel composed of?

A

Different concentrations of acrylamide and a cross-linker, forming a solid, porous matrix

21
Q

What is the first step in GE?

A

Prepare stacking gel (large pores, made with Tris buffer 2 pH units below that of electrophoresis buffer) and resolving/separating gel (smaller pores, pH ~8.8, proteins separated by size)

22
Q

What does the buffer contain in GE?

A

Glycerol, bromophenol and SDS + molecular weight standard proteins

23
Q

What follows electrophoresis in GE?

A

Stain polyacrylamide gel to view protein bands

24
Q

What is a gradient gel?

A

Rather than uniform pore size throughout the gel a gradient is formed by varying acrylamide concentrations

25
Q

What do gradient gels allow for?

A

Separation of a greater range of molecular weights

26
Q

What is SDS?

A

An anionic detergent, which when added to protein sample denatures secondary and tertiary structures

27
Q

What is the resulting molecule following SDS treatment?

A

Linear with overall negative charge

28
Q

What weight ratio do most proteins bind SDS at?

A

1.4 g SDS per gram of protein

29
Q

Where do linear proteins with a negative charge move towards in an electric field?

A

Positive electrode (anode)

30
Q

How can disulphide bonds between subunits of a multi-subunit protein be denatured?

A

With a reducing agent, e.g. 2-ME or DTT

31
Q

In what cases do we need ‘non-reducing’ conditions?

A

Antibodies, to preserve the 3D structure

32
Q

What are the limitations of non-dissociating PAGE? (x2)

A
  1. Proteins of similar molecular size may migrate at different rates due to differences in folding
  2. Folded proteins may be too bulky to move through pores
33
Q

What does the slope in a Ferguson plot represent?

A

The molecular weight

34
Q

What is the concept of 2D gel electrophoresis?

A

Separates proteins first by charge and then by molecular weight

35
Q

What is meant by isoelectric focusing?

A

Proteins migrate in the presence of a continuous pH gradient until the reach their isoelectric point

36
Q

What happens to proteins at their isoelectric point?

A

They migrate at a steady state and become focused into narrow zones

37
Q

What gradients are used in isoelectric focusing?

A

Immobilised pH Gradients (IPGs), which are standardised acrylamide strips with pH gradient co-polymerised within the gel matrix

38
Q

How are IPGs rehydrated?

A

In rehydration buffer containing:

  • a non-ionic detergent (e.g. CHAPS - disrupts hydrophobic interactions)
  • urea (disrupts H-bonds, increases solubility)
  • Reducing agent
  • Carrier ampholytes
39
Q

What voltage is required for IEF?

A

High voltage

40
Q

What is the purpose of the two Tris-based SDS buffers in the 2nd dimension?

A

1st buffer contains DTT (reduces sulphide bonds)

2nd buffer contains iodacetamide (an alkylating agent that prevents the reformation of disulphide bonds)

41
Q

How can the number of proteins to be resolved be reduced? (x3)

A
  1. Subcellular fragmentation (into nuclear, mitochondrial and cytosolic fragments)
  2. Partial purification
  3. Detergent pre-fractionation
42
Q

What is agarose gel electrophoresis (AGE) commonly used in?

A

Separation of DNA fragments

MADD (5 decks)

Brainscape helps you reach your goals faster, through stronger study habits.
© 2024 Bold Learning Solutions. Terms and Conditions