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Fundamentals Of Pathology > Electrophoresis > Flashcards

Electrophoresis Flashcards

1
Q

What is the isoelectric point?

A

Where the protein has no net charge at that pH

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2
Q

What is the charge if pH

A

Net positive charge

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3
Q

What is the charge if pH>pI?

A

Net negative charge

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4
Q

What is pI?

A

Isoelectric point

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5
Q

What are the principles of electrophoresis?

A

Migration of charged particles in the electric field

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6
Q

What is electrophoresis useful for?

A

Separating/ Purifying macromolecules

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7
Q

What does migration of charges depend on in electrophoresis?

A

Charge, size or shape

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8
Q

What gel does the horizontal electrophoresis apparatus normally use?

A

Agarose

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9
Q

What gel does the vertical electrophoresis apparatus normally use?

A

Polyacrylamide

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10
Q

What is the function of the tank in electrophoresis?

A

Where the sample and buffer are attached to a power block

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11
Q

What is the function of the power block in electrophoresis?

A

Supplies electric current through the buffer

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12
Q

What is the function of the casting tray in electrophoresis?

A

Preparing the gel

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13
Q

What is the function of the sample comb in electrophoresis?

A

Makes an indentation in the gel that allows you to put your sample in the buffer before applying the current

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14
Q

What do you need to check when choosing apparatus (3)?

A

Uniform electric field across gel
Cooling to present thermal artefact
Access to gel loading an monitoring

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15
Q

Where is gel electrophoresis usually cast?

A

In a thin slab within wells

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16
Q

What does the buffer provide in gel electrophoresis (3)?

A
  • ions to carry current
  • relatively constant pH
  • pH of solution and nature of R-groups have an important effect on migration of proteins
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17
Q

What is agarose made up of?

A

Polysaccharide extract from seaweed

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18
Q

How is agarose prepared?

A

Dissolving powdered agarose into buffer. Heat in microwave and pour into a casting tray.
Undergoes polymerisation when cooled

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19
Q

Why does agarose have a low resolving power?

A

Big pores

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20
Q

What is agarose used to separate (specifically what size)?

A

Large proteins >200kDa

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21
Q

What concentration is agarose used at?

A

0.5-2%

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22
Q

What is polyacrylamide formed from?

A

The small synthetic molecule acrylamide

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23
Q

What does acrylamide need to be in the presence of to polymerise?

A

Catalyst and initiator (APS and TEMED)

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24
Q

What is the pore size of acrylamide determined by?

A

Polyacrylamide concentration

25
Q

What does TEMED stand for?

A

Tetra methyl ethylene diamine

26
Q

What is the most popular technique for protein electrophoresis?

A

Vertical slab gel

27
Q

What do buffers do in electrophoresis (3)?

A

Supply current carrying ions
Maintains desired pH
Provides medium for heat dissipation

28
Q

What are the two classifications for buffers?

A

Continuous

Discontinuous

29
Q

What is a continuous buffer?

A

Uses same buffer in gel, sample and tank

30
Q

What is a discontinuous buffer (4)?

A

Different buffer
Non-restrictive large-pore gel
Resolving gel -greater restriction
Have different buffers for stacking gel, resolving gel and tank buffer

31
Q

What does protein electrophoresis depend on?

A

The migration of any protein in electricity field depends on pI and pH

32
Q

What happens to the pI of any given protein?

A

Constant

33
Q

What does the pH of a solution determine in protein electrophoresis?

A

The charge expressed by the protein

34
Q

What does SDS-PAGE stand for?

A

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis

35
Q

What type of detergent does SDS-PAGE have?

A

Strong anionic

36
Q

Why is the strong anionic detergent important in SDS-PAGE?

A
  • to solubilise, dissociate and denature most proteins to single polypeptide chains
37
Q

What does SDS-PAGE do to hydrogen bonds?

A

Disrupts them

38
Q

What does SDS-PAGE do to hydrophobic interactions?

A

Blocks them

39
Q

To what ratio of SDS: protein does SDS-PAGE bind?

A

1.4:1

40
Q

What cleaving agents does SDS-PAGE include (with example)?

A

Disulfide bond (beta-mercapto ethanol)

41
Q

What is the migration of the protein determined by in SDS-PAGE?

A

Weight

42
Q

Where do negatively charged molecules migrate towards in SDS-PAGE?

A

Positive pole / anode

43
Q

What determines the effective separation range of SDS-PAGE?

A

Conc of gel

44
Q

What is SDS-PAGE not suitable for?

A

Small polypeptides and peptides with a molecular weight of less than 10kDa

45
Q

What type of buffer system can be used in protein gel electrophoresis?

A

Either (continuous or discontinuous)

46
Q

What are the three protein stains you can use?

A

Coomassie brilliant blue
Fluorescent stain
silver stain

47
Q

How does the fluorescent stain work?

A

Use a fluorescent light to see the samples

48
Q

What are the ratios of coomassie brilliant Blue needed in electrophoresis?

A

9:9:2 v/v/v

Methanol, distilled water and acetic acid

49
Q

How do you compare your sample to known proteins in protein electrophoresis?

A

You have a band of known proteins running down the side

50
Q

When is native gel electrophoresis used?

A

Mainly in circumstances where native conformations are to be analysed (as you don’t have to denature the proteins for it to work)

51
Q

What is the separation based on in native gel electrophoresis (2)?

A

Charge: size ratio and conformation (shape)

52
Q

Give the advantages of native gel electrophoresis (3)

A
  • Potential of separating proteins of identical molecular weight not resolved with SDS-PAGE
  • recovery of protein in native state
  • study binding events (protein- protein or protein- ligand)
53
Q

What are the types of nature gels?

A

Agarose and polyacrylamide

54
Q

What does SPEP stand for?

A

Serum protein electrophoresis

55
Q

What does SPEP measure?

A

Specific proteins in blood

56
Q

How does SPEP work?

A

Uses an electrical field to separate proteins into groups of a similar size, shape and charge

57
Q

What are the two main groups in blood serum?

A

Albumin

Globulins

58
Q

What is a densitometer?

A

Used to scan separated proteins in the gel- the patterns give info about protein fractions

59
Q

What is the pH range of haemoglobin electrophoresis?

A

8-9 (so slightly alkaline)

Fundamentals Of Pathology (20 decks)

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