Electrophoresis Flashcards
What is electrophoresis?
A separation technique based on the migration of charged solutes/particles in a liquid medium under the influence of an electric field
What can electrophoresis be used to isolate and analyse?
proteins, amino acids, nucleic acids
an electrophoretogram is generated
How are the zones visualised?
with an analyte-specific stain and can be quantified using for example a densitometer
What is the principle of electrophoresis?
A constant voltage/current between 2 electrodes creates an electric field that cause the movement of charged ions
What is the anode?
Positive electrode that attracts negatively chared anions
What is the cathode?
Negative electrode that attracts positively charged cations
What is the purpose of the support medium?
Provides the connection between the two electrodes, submerged in an aqueous buffer that carries the current
What does electrophoretic mobility depend on?
physical characteristucs and the method of electrophoresis
What are the basic components of the electrophoretic system?
-Support media
-Power supply
-Electrodes
-Buffer resevoir/chamber
-Visualisation support
and can link to
-Densitometer
What is the support medium usually and what are types of support media?
Usually a crosslinked polymer whose composition and porosity is chosen based on the target analyte
-porous insoluble gels (gel electrophoresis)
-inert membranes (cellulose acetate)
-pure buffer solutions (capillary electrophoresis)
What are the 2 kinds of gels that could be used in gel electrophoresis?
Agarose gels
Polyacrylamide gels
Characteristics of agarose gel?
-Linear polysaccharide polymer made of repeating units of agarobiose (D-galactose and 3,6,anhydro-L-galactopyranose)
-Large range of separation but relatively low resolving power (100-50,000bp DNA)
-Powdered agarose is mixed with buffer to make a gel matrix which contains pores (0.5-2%)
Characteristics of Polyacrylamide gels?
-Polymer prepared by heatinmg acrylamide with a catalyst with or without crosslinking agents
-Thermostable, transparent, relatively chemically inert
-Analytes are separated based on charge : mass ratio and molecular size
-Low range of separation (e.g DNA less than 500bp) but high resolving power - resolves DNA that differ by as little as 2% in length
Inert membranes: Cellulose Acetate - Characteristics
-Membranes are dry, brittle
-Made by treating cellulose with acetate anhydride
-Need to be soaked in buffer to soften before use
-Seldom used in routine clinical applications
Buffer solutions: Capillary Electrophoresis - Characteristics
-Separation carried out in a small-bore fused silica capillary tube containing a buffer/electrolyte solution
-Capillary tube serves as the electrophoretic chamber - connected to a detector and via buffer reservoirs, to a high voltage power supply
-Separation based on size:charge ratio
-All ions move in same direction by electroosmotic flow
-pH of buffer impacts the charge on components in the sample
What are the advantages of CE (capillary electrophoresis)
-Enhanced separation efficiency and reduced separation time
-High throughput - lower handling errors
-Small sample volumes analysed (pL/nL range)
What is the purpose of the power supply?
-Supplies electrical power
-High voltage improves resolution and decreases time needed for separation
-Voltage cannot be increased too much or excessive heating will occur
What is the purpose of the buffers?
-Carries the applied current and establishes the pH
-Ionic strength influences - conductance of the support, rate of molecule migration, sharpness of electrophoretic zones
-High ionic strength buffers yield sharp bands but produce more joule heat, which can affect heat labile proteins
What factors affect the migration rate of molecules?
-Molecular weights, size, molecular shape
-Buffer pH
-Supporting media
-Temp and electrical voltage
-Migration time
What are the types of electrophoresis?
-Conventional Electrophoresis e.g agarose gel electrophoresis
-Non conventional electrophoresis e.g capillary electrophoresis
What are the 3 variations of conventional electrophoresis?
(alter composition of coating gels)
-Isoelectric focusing (IEF)
-Rocket immunoelectrophoresis (RIE)
-SDS-Polyacrylamide gel electrophoresis (PAGE)
What happens in Isoelectric Focusing?
-Use a pH gradient to separate based on isoelectric point
-Gradient is formed using low-molecular weight ampholytes with a range of pI values (e.g pH3-10) -. migrate to establish a pH zone
-Molecules in a mixture migrate until their isoelectric point mtches the local pH - net charge is zero
-Isoelectric focusing can resolve proteins with pI differences as small as 0.01 - high resolution
What does immunoelectrophoresis use?
-Methods use antibody-impregnated agarose gels at high pH (~8.5) to quantitate proteins
What happens in rocket immunoelectrophoresis?
-Samples applied at cathode (antigen excess)
-As protein migrates conc decrease as large stable immune complexes form which precipitate and can no longer migrate
-Sharp arc of precipitated antigen-antibody complexes that resemble a rocket are formed
-Height of the precipitation arc is proprtional to the conc of protein in sample
