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Analytical and Clinical > Week 6 - Electrophoresis > Flashcards

Week 6 - Electrophoresis Flashcards

1
Q

How to identify heavy molecules?

A

moves slower

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2
Q

How to identify small molecules?

A

moves more fast and are more charged

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3
Q

Ohm’s law

A

V/I = R
V=voltage I=current R=resistance

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4
Q

V/I = R

A

V=voltage I=current R=resistance

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5
Q

What is the medium used in electrophoresis?

A

agarose, polyacrylamide

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6
Q

What is the capillary used in electrophoresis?

A

pore size, ionic strength

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7
Q

What determines mobility in electrophorsis?

A

speed of migration

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8
Q

What happens if we apply constant voltage?

A

current will eventually increase

because resistance of medium is decreasing

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9
Q

What is agarose gel used for?

A

DNA

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10
Q

What is polyacrylamide gel used for?

A

high resolution, proteins and smaller
nucleic acid.

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11
Q

Whats the difference between polyacrylamide and agarose?

A

smaller pore size of polyacrylamide compared to agarose
- better separation of small molecules

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12
Q

How can capillary achieve high resolution separation?

A

narrow capillary with buffer

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13
Q

APPLICATIONS OF ELECTROPHORESIS

A

DNA fingerprinting

DNA sequencing

Protein analysis

Clinical diagnosis – serum proteins, biomarkers

Pharmaceutical development – characterisation of biologics e.g.
antibodies, vaccines

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14
Q

What does higher concentration of agarose mean?

A

smaller mesh
- more agarose = smaller the sieve

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15
Q

agarose structure

A

Horizontal

Native structure

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16
Q

What is commonly used at a loading dye in agarose gel?

A

bromophenol blue, xylene cyano

made also with glycerol

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17
Q

What type of staining is used in agarose gel?

A

Ethidium bromide, non-toxic fluorescent stains

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18
Q

Ethidium bromide

A

carcinogenic

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19
Q

What can be determined from agarose gl electrophoresis?

A
  • Size determination
  • DNA or RNA extraction
  • DNA and RNA
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20
Q

Feature of agarose gel

A

we can physically cut out band and re-examine

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21
Q

What is a southern blot?

A

a laboratory technique used to detect specific DNA sequences in a DNA sample

  • the samples are chewed up using DNA restriction enzymes normally in intracellular membrane
22
Q

How to set up a southern blot?

A

use paper to blot and wick up buffer

start to more liquid/buffer up towards the well so we have gel on top

transfer the membrane on top

put something heavy on it

= we have cellulose membrane with DNA transferred

23
Q

How do you use a radio cable P32 probe?

A

incubate membrane with probe

wash off excess

all of prob hydrolyse to particular fragment of DNA that we transferred to membrane

expose to x-ray film

shows restriction image

24
Q

What does SDS-PAGE involve?

A

removing tertiary structure

denatured protein (SDS, β mercaptoethanol, boiling)

discontinuous buffer system

25
Q

SDS

A

sodium dodecyl sulphate

26
Q

PAGE

A

polyacrylamide gel electrophoresis

27
Q

What gel does SDS-PAGE use?

A

5%-20% polyacrylamide

28
Q

What way do samples run on SDS-PAGE?

A

Vertical

29
Q

Where does SDS-PAGE migrate to?

A

towards positive electrode

30
Q

What is the mythology of SDS?

A

disrupted bonds

31
Q

What type of reaction is making polyarcylamines?

A

polymerisation reaction

32
Q

What is the loading dye for SDS-PAGE?

A

Bromophenol blue

33
Q

What is the staining for SDS-PAGE?

A

Coomassie blue, non-
toxic fluorescent stains

34
Q

What does SDS-PAGE determine?

A

size and protein extraction

35
Q

Why does DBS have different pH between stacking gel and resolving gel?

A

allows to load off samples and all out samples are ready and lined up so they can migrate

36
Q

What is the buffer placement like in DBS?

A

buffer at top and bottom

37
Q

What are the other types of gels for SDS?

A

Non-denaturing gels/native gels – separation, identification of protein of interest by substrate

Gradient gels – for more complex mixtures, separation of similar Mr values

Isoelectric focussing gels – based on pI (iso-electric points)

2D PAGE gels

38
Q

What does non-denaturing gels/native gels allow?

A

separation, identification of protein of interest by substrate

39
Q

What do gradient gels allow?

A

for more complex mixtures, separation of similar Mr values

40
Q

What are Isoelectric focussing gels based on?

A

based on pI (iso-electric points)

41
Q

What are the advantages of gradient gel?

A

more specific gel creation with different pore sizes and concentration
and smaller molecules are going to percolate further

42
Q

Describe the molecule distribution in gradient gels.

A

smaller molecules are going to percolate further

larger molecules are going to be stuck at the top because they can’t pass through the sieve

different sizes of molecules are going down

43
Q

What is immunoblotting/western blotting?

A

Transfer of products to a solid stable membrane – nitrocellulose

Further investigate products – antibodies

44
Q

Process of protein: antibody detection

A
  1. coat surface with sample (antigen)
  2. block unoccupied sites with non-specific proteins
  3. incubate with primary antibodies against specific antigens
  4. incubate with primary antibody against specific antigen
  5. add substrate
  6. formation of coloured products indicates presence of specific antigen
45
Q

What does isoelectric focussing tell us?

A

isoelectric points of protein

46
Q

Process of isoelectric focussing

A

a protein sample may be applied to one end of a gel strip with an immobilised pH gradient.
or a protein sample in a solution of ampholtes maybe used to rehydrate a dehydrated gel strip

an electric field is applied

after staining, proteins are shown to be distributed along pH gradient according to their pI values

47
Q

What happened if the spot is at the positive side in the isoelectric focussing?

A

lower pI - lower pH

48
Q

What happened if the spot is at the negative side in the isoelectric focussing?

A

higher pI and higher pH

49
Q

What happens if the pH is the same as pI?

A

netcharge = 0

50
Q

What does 2D electrophoresis allow?

A

allows a 2 way separation

51
Q

What are the two separation proteins in the two dimensions?

A

1D
= using isoelectric focusing

2D
= SDS-PAGE gel

52
Q

What does capillary electrophoresis allow?

A

checking purity

Analytical and Clinical (11 decks)

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