Principles of Electrophoresis Flashcards
What is the principle of electrophoresis
It’s a separation technique based on the migration of charged solutes/particles in a liquid medium under influence of an electrical field
Electrophoretic mobility
Dissimilar molecules in a mixture will separate as they move at different rates - size influences, bigger molecules will move slower
What can electrophoresis be used to isolate & analyse
- Proteins
- Amino acids
- Nucleic acids
What is generated
An electrophoretogram
What are zones visualised with
An analyte specific stain - e.g: Sybr Green
Function of the electrodes
Constant voltage/current between the 2 electrodes creates an electric field that causes movement of charged ions
Anode
Pos electrode that attracts neg charged anions
Cathode
Neg electrode that attracts pos charged cations
Function of the support medium
Provides the connection between the 2 electrodes
What does the electrophoretic mobility depend on
- Physical characteristics
- Method of electrophoresis
3 types of support media
- Porous insoluble gels (gel electrophoresis)
- Inert membranes (cellulose acetate)
- Pure buffer solutions (capillary electrophoresis)
What is agarose
Linear polysaccharide polymer made of repeating units of agarobiose
Pro of agarose gel
Large range of separation
Con of agarose gel
Low resolving power
What is polyacrylamide & how is it prepared
A polymer, prepared by heating acrylamide with a catalyst, with or without crosslinking agents
3 characteristics of polyacrylamide gels
- thermostable
- transparent
- relatively chemically inert
What are analytes separated based on with polyacrylamide gel
Based on charge
Pro & Con of polyacrylamide gel
Pro: High resolving power (can resolve DNA that differs by as little as 2% in length)
Con: Low range of separation
How are the inert membranes, cellulose acetate, made
Made by treating cellulose with acetic anhydride
What do the cellulose acetate membranes need to be soaked in before use
Buffer
What is separation based on in capillary electrophoresis
Separation based on size to charge ratio
in capillary electrophoresis, what direction do all ions move in & what causes this
In the same direction by electro-osmotic flow
Advantages of capillary electrophoresis
- Enhanced separation efficiency
- Reduced separation time
- Lower handling errors
- Small sample volumes analysed
System components of capillary electrophoresis
- Power supply
- Buffers
What does high voltage improve in capillary electrophoresis
Improves resolution & decreases time needed for separation
Function of buffers in capillary electrophoresis
Carries the applied current & establishes the pH
What do high ionic strength buffers yield? what do these produce and then affect/
High ionic strength buffers yield sharp bands.
But these produce more Joule heat.
This can affect heat labile proteins.
What are the factors affecting migration rates of molecules
- Molecular weight, size & shape
- Buffer pH
- Supporting media
- Temp
- Electrical voltage
- Migration time
What are the 3 major variations possible of conventional electrophoresis (agarose gel electrophoresis)
- Isoelectric focusing (IEF)
- Rocket immunoelectrophoresis (RIE)
- SDS-Polyacrylamide gel electrophoresis (PAGE)
What is used to separate bands in Isoelectric Focusing and what is the separation based on
Isoelectric focusing uses a pH gradient to separate, based on isoelectric point
In isoelectric focusing, how is gradient formed
Gradient is formed using low molecular weight ampholytes with a range of pI values
In isoelectric focusing, what do molecules in a mixture migrate until
The molecules in the mixture migrate until their isoelectric point matches the local pH (net charge is then 0)
Principle of immunoelectrophoresis
Use of antibody impregnated agarose gel at high pH to quantitate proteins
How does rocket immunoelectrophoresis work
- Samples applied at cathode, where antigen is in excess
- As proteins migrate, the concentration decreases
- As large stable immune complexes form, these precipitate & can no longer migrate
- Sharp arc of precipitated antigen-antibody complexes that resemble rocket
- Height of precipitation arc is proportional to con of protein in sample
What is the height of the precipitation arc proportional to
Height of precipitation arc is proportional to concentration of protein in sample
What is the difference in dissociating vs non-dissociating PAGE (polyacrylamide gel electrophoresis)
With (SDS-PAGE) or without (nondenaturing PAGE, no SDS)
SDS = Sodium Dodecyl Sulfate
What is the difference between continuous & discontinuous PAGE
- Continuous: uses a single gel that acts as a molecular sieve with the same buffer ions in sample and gel5
- Discontinuous: uses 2 gel systems-> Stacking (large pores) and resolving (small pores) with different buffers in gel compared to buffer reservoir
Clinical application of serum protein electrophoresis
Separates serum proteins into 5 major bands - albumin, alpha 1 globulins, alpha 2 globulins , beta globulins & gamma globulins
What type of data do electrophoretograms give
Qualitative & quantitative
In serum protein electrophoresis, which protein band is increased in multiple myeloma
The gamma band
Principle of Hb electrophoresis
Haemolysed blood sample analysed & Hb content separated
Separation of normal proteins & detection of Hb variants e
Examples of isoenzymes that can be separated & quantitated electrophoretically
Creatine kinase (CK) & lactate dehydrogenase (LD)
Separation of isoenzymes steps
- After separation, substrates added
- Rxn occurs leading to product that is detected
What can isoenzyme separation provide info on
Organ damage - different isoenzymes produced by different organs
