Electrophoretic Techniques Flashcards
What is the principle of Electrophoresis?
Migration of a charged particle in an electric field
What THREE characteristics are related to Electrophoretic Mobility, μep?
- Net electric charge, q
- Molecular size / mass, f
- Molecular shape, f
What are the FOUR advantages of Electrophoresis?
- High Resolution
- High Sensitivity
- Multiplicity
- Simple Set Up
What TWO basic electrical equations are important in Electrophoresis?
- Ohm’s Law: I = E/R
- Power/Heat Generation: P = EI
TRUE OR FALSE:
Only one electrical parameter is held constant in Electrophoresis?
True
What do fast Electrophoresis separations require?
High V or I
* create a lot of heat
What do high temperatures cause DURING Electrophoresis?
- Denaturation of proteins
- Smile Effect
What TWO types of gel are used in VERTICAL Electrophoresis systems?
- Rods
- Slabs
What type of gel is used in HORIZONTAL Electrophoresis systems?
Agarose
What is the role of the gel matrix in EP?
Inhibits convection and diffusion which would impede separation of molecules
What is Agarbiose?
Repeating units of agarose
* Galactose
* Anhydrogalactose
What concentration is agarose gel used at in EP?
0.7 - 2% w/w
What do lower concentrations of agarose gel cause?
Larger pores
What does this Ferguson Plot indicate about the protein?
- Same charge densities
- Separation by size
What does this Ferguson Plot indicate about the protein?
Protein with higher charge density has smaller size
What does this Ferguson Plot indicate about the protein?
Large protein has higher free mobility
What does this Ferguson Plot indicate about the protein?
Same size but different mobilities
What does SDS do to the protein in EP?
Coats the protein in a NEGATIVE charge
What are the THREE parameters of EP separations?
- SEPARATION - distance between bands
- RESOLUTION - separation relative to band width
- SHARPNESS
What are the FIVE factors affecting band sharpness in EP?
- Sample Volume
- Sample Ion Concentration
- Diffusion Effects
- Gel Density
- Mode
What are the THREE characteristics of the Stacking Gel for DISC PAGE?
- Large pore size
- Low pH (Tris-HCl)
- Concentrates into ~20 μm zone
What are the THREE characteristics of the Resolving Gel for DISC PAGE?
- Higher Density
- High pH (Tris-Glycine)
- Separation
In isoelectrofocusing, what is used to produce gels with pH gradients?
Ampholytes
What technique is used for whole cell proteome analysis?
2D Electrophoresis
What does the primary antibody do in Western Blotting?
Raised against protein of interest
What does the secondary antibody do in Western Blotting?
Recognises primary antibody
What label catalyses CL reaction with luminol, enhancer and H2O2?
HRP
What is the wavelength of the RED channel in Western Blotting?
680 nm
What is the wavelength of the GREEN channel in Western Blotting?
800 nm
Which channel are protein markers detected in?
Red channel
