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Pathology 1 > Electrophoresis > Flashcards

Electrophoresis Flashcards

1
Q

What are the building blocks of proteins?

A

Amino acid residues

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2
Q

Describe gel electrophoresis

A
  1. Gel cast in a thin slab shape with wells
  2. Immersed within a buffer
  3. Proteins separated within a gel in a series of pores
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3
Q

What are the different detection methods used for electrophoresis?

A

Fluorescent stain
Silver stain
Protein staining (in situ)
- coomassive brilliant blue dyes used as 0.1% (w/v) in
methanol, distilled H2O + acetic acid (9:9:2)

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4
Q

What is the significance of the buffer in gel electrophoresis?

A

Provides ions to carry the current
Maintains a relatively constant pH
The pH of the solution and nature of amino acids R groups have significant effect on protein migration

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5
Q

In basic conditions, what is the overall protein charge?

A

net -ve charge due to COO- carboxyl group

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6
Q

What is the distance migrated by proteins in electrophoresis determined by?

A

Migration of protein not determined by intrinsic electric charge but by weight

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7
Q

What is the role of SDS-PAGE’s strong anionic detergent?

A

Solubilises dissociate and denature most proteins to single polypeptide chains
Disrupts H+ bonds
Blocks hydrophobic interactions
- binds at ratio of 1.4g of SDSg-1 of protien conferring net negative charge to polypeptide in proportion to length

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8
Q

What are the different types of electrophoretic gel?

A
  1. Agarose

2. Polyacrylamide

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9
Q

Which amino acid group determines the charge of the amino acid?

A

R group determines if the amino acid is neutral, acidic or basic
The protein backbone has no charge

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10
Q

What is protein electrophoresis?

A

Migration of any protein in an electric field depending on pI and pH

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11
Q

What is a discontinuous buffer system?

A

Uses different buffers

  • > non restrictive large pore gel
  • > resolving gel - greater resolution
  • > have different buffers for stacking gel ,resolving gel, and tank buffer
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12
Q

What is the pI of proteins?

A

pI is constant for any given protein

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13
Q

What is electrophoresis?

A

The migration of charged macromolecules in an electric field

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14
Q

What is the significance of pH in protein electrophoresis?

A

pH of solution determines the charge of protein

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15
Q

When is Native non denaturing gel electrophoresis?

A

Used when native conformational need analysing

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16
Q

What is SDS-PAGE?

A

Most commonly used electrophoretic technique for separation containing disulfide bond cleavage agents e.g. β-mercaptoethanol

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17
Q

How is Agarose gel formed?

A
  • polysaccharide extract from seaweed
  • prepared by dissolving powdered agarose in buffer
  • heated and poured into casting tray
  • polymerased when cooled
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18
Q

Outline the main features of native gel electrophoresis

A
  • Biological activity of proteins preserved (not denatured)
  • Polyacrylamide / agarose used
  • Runs without SDS
  • Proteins separated by size, charge and shape
  • Separates proteins even with same molecular weight
19
Q

What is electrophoresis migration based on?

A
  • Current
  • Size
  • Shape
  • Charge
  • Resistance
20
Q

What is the use of agarose gel?

A

Has a relatively low resolving power as pores larger

used to separate large proteins >200KDa at a [0.5-2%]

21
Q

Outline the features of Denaturing SDS-PAGE gel electrophoresis

A
  • Negatively charged, strong anionic detergent SDS
  • SDS denatures and dissociates proteins into polypeptide chains
  • SDS complexes with proteins to mask charge
  • Separtion based on molecular size only
22
Q

What is a continuous buffer system?

A

Buffer system using same buffer in gel, the sample and tank

23
Q

What is the net charge of proteins in acidic buffer conditions

A

net +ve charge due to NH3+

24
Q

Describe Hb electrophoresis

A

pH range of 8-9 (alkaline)

majority proteins will be negatively charged

25
Q

What are the features of Native gel electrophoresis?

A

Run without SDS
Proteins aren’t denatured
Separation based on charge:Size ratio and shape
Charge changes with buffer pH changes

26
Q

How are the separated proteins detected in SPEP?

A

A densitometer is used to scan separated proteins in the gel pattern shows protein fractions

27
Q

What is the use of electrophoresis?

A

Useful for separating/purifying macromolecules consisting of many sub units with multiple ionisable charged groups

28
Q

What are the 2 major protein groups in blood serum?

A
  • Albumin

- Globulins

29
Q

What are the 2 apparatus types of electrophoresis?

A
  1. Horizontal - usually for agarose gel

2. Vertical - for polyacrylamide gel

30
Q

What do we have to ensure when choosing type of electrophoresis apparatus?

A
  • Electric field is uniform across gel
  • To cool to prevent thermal artefacts
  • Is access to gel monitoring and loading
31
Q

What is the pI?

A

pI - isoelectric point

Zwitterions, when protein has no net charge at a certain pH neutral due to COO- and NH3+

32
Q

What is blood composed of?

A

Blood composed of cells and plasma made up of water, proteins, salts, glucose, hormones and clotting factors

33
Q

What is the polyacrylamide gel’s pore size determined by?

A

Polyacrylamide has smaller pores than agarose determined by [polyacrylamide]

34
Q

What are buffer systems classified as?

A
  • Continuous

- Discontinuous

35
Q

What is the charge of proteins?

A

Proteins can either have a net -ve or +ve charge dependent on the pH of the buffer

36
Q

What are the benefits of native gel electrophoresis?

A
  • Can separate proteins with identical molecular weight
  • Proteins can be recovered in native state
  • Can steady binding
37
Q

How is polyacrylamide gel formed?

A

Formed from synthetic small molecule acrylamide
- polymerises into long chains in presence of APS & TEMED
APS = catalyst
TEMED = initiator

38
Q

What are the clinical applications of native gel electrophoresis?

A

Can measure specific proteins in blood
SPEP (serum protein electrophoresis) uses an electric field to separate proteins into groups of similar size, shape and charge helps identify disease

39
Q

What is the significance of [gel]?

A

[gel] determines effective separation range of SDS-PAGE
SDS-PAGE not suitable for small peptides of molecular weight <10KDa
- dis/continuous buffer system can be used in protein gel electrophoresis

40
Q

Describe the macromolecules in electrophoretic gel

A

In an electric field, negatively charged molecules migrate towards positive pole

41
Q

What are the types of gel used in native electrophoresis?

A

Agarose & Polyacrylamide

42
Q

What is the role of buffers?

A

Supplies current carrying ions in electrophoretic cell
Maintains desired pH
Provides a medium for heat dissipation

43
Q

Describe the structure of amino acids

A

2 ionisable groups
- carboxyl group terminus
- amino group terminus
Variable side chain (R group)

44
Q

What is the most common technique for protein electrophoresis?

A

Vertical slab gel electrophoresis (polyacrylamide)

Pathology 1 (20 decks)

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