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BGM1002-Biochemistry > BGM1002/L09 Gel Electrophoresis > Flashcards

BGM1002/L09 Gel Electrophoresis Flashcards

1
Q

Define gel electrophoresis.

A

Movement of dispersed particles relative to a fluid under the influence of a spatially uniform electric field

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2
Q

What does pore size depend on in gel electrophoresis?

A

Concentration of gelling agent

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3
Q

What is agarose?

A

Linear polymer extracted from red seaweed

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4
Q

What is acrylamide?

A

Organic compound with ultimate functional groups

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5
Q

Why does the liquid phase need to be buffered in gel electrophoresis?

A

To prevent current-induced pH changes and to minimise heating

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6
Q

Name 3 factors that affect gel electrophoresis.

A

Net charge of molecule
Size of molecule
Electric field strength
Properties of gel
Properties of running buffer
Temperature

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7
Q

What is PAGE?

A

Polyacrydamide gel electrophoresis

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8
Q

What is SDS-PAGE?

A

Gel electrophoresis based on protein size in which protein is denatured with heat/SDS

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9
Q

What biological molecule can be ran on PAGE gels?

A

DNA

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10
Q

What can PAGE gels do?

A

Oxidise and produce non-native disulfide bonds

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11
Q

What is SDS-PAGE 2?

A

Discontinuous gel electrophoresis made of 2 gels - stacking and resolving gels

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12
Q

Give 3 properties of stacking gels.

A

pH 6.5
Focuses proteins before they’re separated
Low % acrylamide

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13
Q

Give 3 properties of resolving gels.

A

Separates proteins
Higher % acrylamide
Changing affects resolution

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14
Q

Name the 3 buffer components in SDS-PAGE 2.

A

Glycine/chloride counter ions
SDS - to ensure protein denatures
Tris buffer - pH 8.3

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15
Q

What are gradient gels?

A

Gels in which acrylamide concentration increases down the gel

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16
Q

What concentration of agarose is in TAE/TBE buffer?

A

0.5-2%

17
Q

Give the 3 buffer components in agarose gel electrophoresis.

A

Tris - pH 8.3
Acetate/borate counter ions
EDTA - chelating agent

18
Q

What separation range does agarose gel electrophoresis have?

A

50bp-20kbp

19
Q

Which has a higher resolution: agarose gel electrophoresis or PAGE?

A

PAGE

20
Q

Why do DNA and most proteins need to be dyed?

A

They have no intrinsic colour

21
Q

Name an intercalating dye.

A

Ethidium bromide

22
Q

Name 2 minor groove binders.

A

Hoechst
DAPI

23
Q

Name a syanine dye.

A

SYBR

24
Q

What is Rf?

A

Migration distance of protein/migration distance of dye

25
Q

What do modern gel imagers do? (2)

A

Plot log(MW) standards against Rf
Solve equation to determine MW of unknown substances

26
Q

Name 4 common problems with gels.

A

Smileys (voltage too high)
Melting gels (temp too high)
Smearing (overloading)
Contaminants
Bubbles
Speckles
Floaty samples
Leaky gel tanks

27
Q

What is UV/visible spectroscopy affected by? (2)

A

Solvent
Environment

28
Q

Define bathochromic/hypsochromic.

A

Basochromic - shift to longer wavelength
Hypsochromic - shift to shorter wavelength

29
Q

What does UV/visible spectroscopy depend on? (2)

A

Quantification of protein
Protein unfolding
Characterisation of substrate/cofactor binding
Biochemical assays

30
Q

What are the assumptions of the Beer-Lambert-Bouger Law? (2/5)

A

Monochromatic/parallel illumination
Homogenous solution
No scatter from medium
Linear absorbance change with concentration
Linear response of detector

31
Q

At what wavelength do Trp, Tyr and disulfides absorb UV light?

A

280nm

32
Q

What programme can be used to quantify proteins?

A

ProtParam

33
Q

What indirect methods can be used to quantify proteins? (2/4)

A

Lowry
Bradford
Blurted method
BCA assay

34
Q

Describe the blurted method of indirect protein quantification.

A

React protein with copper sulfate, sodium hydroxide and potassium tartrate

35
Q

Describe the BCA assay method of indirect protein quantification.

A

Bind copper to nitrogen in protein; complex bound to bicinchonic

36
Q

Give 2 disadvantages to the blurted method of protein quantification.

A

Slow (20-30 mins for readout)
Not very sensitive (1-20mg)

37
Q

Give 1 advantage and 1 disadvantage to the BCA assay method of protein quantification.

A

Very sensitive (1mg)
Slow (1 hour readout)

BGM1002-Biochemistry (29 decks)

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