BGM1002/L09 Gel Electrophoresis

Question 1

Define gel electrophoresis.

Answer 1

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

Question 2

What does pore size depend on in gel electrophoresis?

Answer 2

Concentration of gelling agent

Question 3

What is agarose?

Answer 3

Linear polymer extracted from red seaweed

Question 4

What is acrylamide?

Answer 4

Organic compound with ultimate functional groups

Question 5

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

Answer 5

To prevent current-induced pH changes and to minimise heating

Question 6

Name 3 factors that affect gel electrophoresis.

Answer 6

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

Question 7

What is PAGE?

Answer 7

Polyacrydamide gel electrophoresis

Question 8

What is SDS-PAGE?

Answer 8

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

Question 9

What biological molecule can be ran on PAGE gels?

Answer 9

DNA

Question 10

What can PAGE gels do?

Answer 10

Oxidise and produce non-native disulfide bonds

Question 11

What is SDS-PAGE 2?

Answer 11

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

Question 12

Give 3 properties of stacking gels.

Answer 12

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

Question 13

Give 3 properties of resolving gels.

Answer 13

Separates proteins
Higher % acrylamide
Changing affects resolution

Question 14

Name the 3 buffer components in SDS-PAGE 2.

Answer 14

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

Question 15

What are gradient gels?

Answer 15

Gels in which acrylamide concentration increases down the gel

Question 16

What concentration of agarose is in TAE/TBE buffer?

Answer 16

0.5-2%

Question 17

Give the 3 buffer components in agarose gel electrophoresis.

Answer 17

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

Question 18

What separation range does agarose gel electrophoresis have?

Answer 18

50bp-20kbp

Question 19

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

Answer 19

PAGE

Question 20

Why do DNA and most proteins need to be dyed?

Answer 20

They have no intrinsic colour

Question 21

Name an intercalating dye.

Answer 21

Ethidium bromide

Question 22

Name 2 minor groove binders.

Answer 22

Hoechst
DAPI

Question 23

Name a syanine dye.

Answer 23

SYBR

Question 24

What is Rf?

Answer 24

Migration distance of protein/migration distance of dye

Question 25

What do modern gel imagers do? (2)

Answer 25

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

Question 26

Name 4 common problems with gels.

Answer 26

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

Question 27

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

Answer 27

Solvent Environment

Question 28

Define bathochromic/hypsochromic.

Answer 28

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

Question 29

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

Answer 29

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

Question 30

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

Answer 30

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

Question 31

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

Answer 31

280nm

Question 32

What programme can be used to quantify proteins?

Answer 32

ProtParam

Question 33

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

Answer 33

Lowry Bradford Blurted method BCA assay

Question 34

Describe the blurted method of indirect protein quantification.

Answer 34

React protein with copper sulfate, sodium hydroxide and potassium tartrate

Question 35

Describe the BCA assay method of indirect protein quantification.

Answer 35

Bind copper to nitrogen in protein; complex bound to bicinchonic

Question 36

Give 2 disadvantages to the blurted method of protein quantification.

Answer 36

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

Question 37

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

Answer 37

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