BGM1002/L09 Gel Electrophoresis Flashcards
Define gel electrophoresis.
Movement of dispersed particles relative to a fluid under the influence of a spatially uniform electric field
What does pore size depend on in gel electrophoresis?
Concentration of gelling agent
What is agarose?
Linear polymer extracted from red seaweed
What is acrylamide?
Organic compound with ultimate functional groups
Why does the liquid phase need to be buffered in gel electrophoresis?
To prevent current-induced pH changes and to minimise heating
Name 3 factors that affect gel electrophoresis.
Net charge of molecule
Size of molecule
Electric field strength
Properties of gel
Properties of running buffer
Temperature
What is PAGE?
Polyacrydamide gel electrophoresis
What is SDS-PAGE?
Gel electrophoresis based on protein size in which protein is denatured with heat/SDS
What biological molecule can be ran on PAGE gels?
DNA
What can PAGE gels do?
Oxidise and produce non-native disulfide bonds
What is SDS-PAGE 2?
Discontinuous gel electrophoresis made of 2 gels - stacking and resolving gels
Give 3 properties of stacking gels.
pH 6.5
Focuses proteins before they’re separated
Low % acrylamide
Give 3 properties of resolving gels.
Separates proteins
Higher % acrylamide
Changing affects resolution
Name the 3 buffer components in SDS-PAGE 2.
Glycine/chloride counter ions
SDS - to ensure protein denatures
Tris buffer - pH 8.3
What are gradient gels?
Gels in which acrylamide concentration increases down the gel
What concentration of agarose is in TAE/TBE buffer?
0.5-2%
Give the 3 buffer components in agarose gel electrophoresis.
Tris - pH 8.3
Acetate/borate counter ions
EDTA - chelating agent
What separation range does agarose gel electrophoresis have?
50bp-20kbp
Which has a higher resolution: agarose gel electrophoresis or PAGE?
PAGE
Why do DNA and most proteins need to be dyed?
They have no intrinsic colour
Name an intercalating dye.
Ethidium bromide
Name 2 minor groove binders.
Hoechst
DAPI
Name a syanine dye.
SYBR
What is Rf?
Migration distance of protein/migration distance of dye
What do modern gel imagers do? (2)
Plot log(MW) standards against Rf
Solve equation to determine MW of unknown substances
Name 4 common problems with gels.
Smileys (voltage too high)
Melting gels (temp too high)
Smearing (overloading)
Contaminants
Bubbles
Speckles
Floaty samples
Leaky gel tanks
What is UV/visible spectroscopy affected by? (2)
Solvent
Environment
Define bathochromic/hypsochromic.
Basochromic - shift to longer wavelength
Hypsochromic - shift to shorter wavelength
What does UV/visible spectroscopy depend on? (2)
Quantification of protein
Protein unfolding
Characterisation of substrate/cofactor binding
Biochemical assays
What are the assumptions of the Beer-Lambert-Bouger Law? (2/5)
Monochromatic/parallel illumination
Homogenous solution
No scatter from medium
Linear absorbance change with concentration
Linear response of detector
At what wavelength do Trp, Tyr and disulfides absorb UV light?
280nm
What programme can be used to quantify proteins?
ProtParam
What indirect methods can be used to quantify proteins? (2/4)
Lowry
Bradford
Blurted method
BCA assay
Describe the blurted method of indirect protein quantification.
React protein with copper sulfate, sodium hydroxide and potassium tartrate
Describe the BCA assay method of indirect protein quantification.
Bind copper to nitrogen in protein; complex bound to bicinchonic
Give 2 disadvantages to the blurted method of protein quantification.
Slow (20-30 mins for readout)
Not very sensitive (1-20mg)
Give 1 advantage and 1 disadvantage to the BCA assay method of protein quantification.
Very sensitive (1mg)
Slow (1 hour readout)
