Electrophoresis Flashcards
what is the principle of electrophoresis
constant voltage/current between two electrodes forms electric field to cause movement of charged ions
Anode: pos, attracts neg anions
Cathode: neg, attracts pos cations
funtion of support medium?
provides connection between two electrodes and is submerged in an aqueous buffer that carries current
what are the types of support media
usually made of crosslinked polymers
- porous insoluble gels (gel electrophoresis)
- inert membranes (cellulose acetate)
- pure buffer solutions (capillary electrophoresis)
write a note on agarose gels
agarose is a linear polysaccharide polymer made of repeating units of agarobiose (D-galactose and 3,6, anydro-L-galactopyrnanose)
It has a large range of separation but low resolving power. pores can be a variety of sizes - 0.5% to 2%
write a note on polyacrylamide gels
are thermostable, transparent, relatively chemically inert. analytes separate based on charge
has a low range of separation but high resolving power
write a note on cellulose acetate
is an inert membrane. membrane is dry and brittle since it was made by treating cellulose with acetic anhydride- so need to be soaked in buffer to soften before use
write a note on buffer solutions - capilary electrophoresis
- separation carried out in a small bore fused silica capillary tube with buffer/electrolyte solution
- capillary tubes serves as the electrophoretic chamber, connected to detector and to a high voltage power supple
what is the principle of CE separation
based on size- charge ratios
- electroosmotic flow causes all ions to move in same direction induced by applied potential
- pH of buffer impacts the charge on components in the sample
what are the advantages of CE
-enhanced separation efficiency and reduced separation time
- high throughput- lower handling errors
- small volumes can be analysed (pL and n/L range
what are the system components of CE
power supply - high voltage improves resolution and decreased separation time
- voltage cannot be in creased too much as so to avoid heating
buffers
- carry applied current and establish pH
- ionic strength influences
1. conductance of the support
2. rate of molecule migration
3. sharpness of electrophoretic zones
what factors affect migration rates of molecules
- molecular weight, size, shape
- buffer pH
- supporting media
- temp and electrial voltage
- migration time
write a note on conventional electrophoresis
variations - three variations possible to alter composition of coating gels
- isoelectric focusing
- rocket immunoelectrophoresis
- SDS - polyacrylamide gel electrophoresis
give example of non conventional electrophoresis
capillary electrophoresis
write a note on isoelectric focusing
- use of pH gradient to separate based on isoelectric points
- gradient is formed using low molecular weight ampholytes with range of pI values which migrate to establish a pH zone
- molecules in mixture migrate until their isoelectric point matches the local pH so the net charge is 0
- isoelectric focusing can resolve proteins with pI differences as small as 0.01
what is immunoelectrophoresis
use of antibody impregnated agarose gels at high pH to quantitate proteins
write a note on rocket immunoelectrophoresis
- samples are applied at cathode
- protein migrates and conc decreases because large stable immune complexes form which precipitate and no longer form
- height of the precipitation arc is proportional to the conc of protein in sample
what are the two types of polyacrylamide gel electrophoresis
- dissociating vs non-dissociating
- SDS-PAGE: linearises polypeptide and gives proteins uniform net charge (neg) in gel of specified pore size- molecular sieving
- nondenaturing PAGE- no SDS: preserves native structure and biological activity, separates based on mass:charge ratio - Continuous Vs discontinuous
- continuous: single gel, sample buffer ions in sample, gel and buffer reservoirs
- discontinuous: 2 gel systems: stacking (large pores) and resolving (small pores) with diff buffer in gel than buffer reservoir.
- initial stacking of components as narrow zone improves the resolution
- molecules of simialr size are separated by charge, molecules of simialr charge ae separated by size
what are the clinical applications of electrophoresis
- serum protein electrophoresis,
- hemoglobin electrophoresis
- haemolysed blood sample can be analysed.
- separation of normal Hb (A1,A2,) and detection of variants: S, D, C, E
HbC- hemolytic anaemia
HbS- sickle cell anaemia
- isoenzyme separation
- i.e creatiine kinase, lactate dehydrogenase
- after separation - substrates added, reaction occurs - product detected
- can provide info on organ damage
