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principle and techniques of electrophoresis (factors affecting rate of…
principle and techniques of electrophoresis
General principle
two electrodes of opposite charge (anode, cathode) connected by a conducting medium called an electrolyte
anode is +ve charge, cathode is -ve charge
electric potential (E) is generated and e- will move from anode to cathode. (+ -> -)
separation based on their differences in their migration speed
under the influenced of E
net charge of molecule
the ionization of amino acids
isoelectric point (pl) - pH value at which the protein has a net charge of zero
isoelectric focusing - a method used to separate or purify a mixture of protein using their isoelectric point (1-D concept)
by using gel
each protein will move towards the direction of the electric force and will stop moving when their net charge is zero/ reach isoelectric point
general operation
power supply
simple power supply can be regulated by the voltage. max output up to 200 V
power supplies of IEF need high voltage
max output up to 3 to 5 kV, 200 mA, 100 W
cooling/ heating thermostatic circulator
it is also means that the power and the current must be reduced if only part of a flatbed gel is used
to maintain the temperature of the system from being too hot or too cold
separation chamber
strength of the electric field
properties of supporting medium
gel matrix
have adjustable and regular pore sizes, be chemically inert and not exhibit electroendosmosis
starch gel
prepared from hydrolized potato starch
the pore size can be adjusted by the starch concentration in the solution
disadvantages - low reproducibility and impractical handling
agrose gel
mostly used when large pores for the analysis of molecule >10 nm in diameter are needed
obtained from red seaweed (polysaccharide)
the pore size depends on the concentration of agarose
dextran gel
solely used for preparative methods wo sieving effect
polyacrylamide gels
chemically innert, fully transparent and mechanically stable
v = mE
the mobility (m) is determined by the size particle, shape, and charge and the temperature during the separation
high current is used, IEF are smaller because the pH gradient has a relatively low conductivity
factors affecting rate of migration (mobility)
strength of the electric field (E)
properties of supporting medium
temperature of operation
effect of pH
net charge of molecule
size and shape of particles
cationic detergent electrophoresis
strongly acid proteins do not bind SDS
is to use cationic detergents for instance, cetyltrimethyl ammonium bromide (CTAB), pH 3-5.
allow separation according to the molecular sizes in the direction of the cathode
the separation pattern is diff from the SDS electrophoresis => the detergent-protein micelles are differently structured
can causes less damage to protein than SDS
buffer
commonly used for 2-D SDS-PAGE is Tris-glycine system
separates proteins at high pH
advantage of minimal protein aggregation
clean separation even at relatively heavy protein loads
Ettan DALT precast gels utilize a buffer system based on piperidinopropionamide (PPA) -> high separation pH of the Laemmli system
Tris-tricine system for improving resolution of polypeptides with molecular weight values below 10000
types of electrophoresis
capillary electrophoresis
for analytical and micropreparative electrophoresis
as for HPLC (high-performance liquid chromatography)
separation is carried out in a fused silica capillary tube 20-30 cm long and with an internal diameter off 50-100 micro meter.
zone electrophoresis
agarose gels with concentration 0.7-1% are often used in the analysis of serum protein
the separation times are extremely low - 30 min
have large pore size
by using agarose gel, analysis of detection of specific protein are very suitable by immunofization. after electrophoresis the specific antibody is allowed to diffused through the gel
in this way only the desired fraction are detected during development
second dimension electrophoresis (SDS-PAGE)
sodium dodecyl Sulfate-Polyacrylamide gel electrophoresis
separates proteins according to their molecular weight
each spot on the resulting 2-D corresponds to a single protein species in the sample.
can obtain:
protein pl
apparent molecular weight
the amount of each prot
principles