factors affecting electrophoretic mobility.ppt

A presentation on Electrophoresis
Presented by Dr. Moyeh Marcel NAHPI uba
Uploaded by Emmarise

1. Factors affecting electrophoretic mobility
a)Net charge on the sample
From the equation
µ= Q/6Πηrv
WhereQisthenetchargeonthesample
Sample net charge is directly proportional to electrophoretic
mobility
Higher the net charge, the faster the sample moves in an electric
field
Net charge affected by pH of medium
pH will either protonate or deprotonate a sample giving it a net
positive, negative or neutral charge hence affecting mobility

2. Factors affecting electrophoretic mobility
b) Particle size and Shape
From the equation
µ= Q/6Πηrv
•Radius(size)oftheparticleisinverselyproportionaltothe
electrophoreticmobility.
Large particles get retarded in the support medium than smaller ones
Large particles also experience more frictional and electrostatic forces
delaying motion
•Shape of the particle also influence µ.
Rounded or spherical particles offer less frictional resistance
compared to sharp edged shaped-particle that adhere to support
medium increasing friction reducing µ

3. Factors affecting electrophoretic mobility
c) Strength of the electric field
The electric field is given by Ohm’s law which states that
I=V/R
An increase in the voltage applied will lead to an increase in the
rate of sample
migration
If the I is increased, migration rate will increase but may also
cause heat production affecting gel viscosity and increasing
convection currents
If the resistance of the system is increased, it will lead to a
decrease in the rate of the migration

4. Factors affecting electrophoretic mobility
d) Properties of support medium
i. Adsorption by support medium
May cause sample trailing and reduction in the rate of migration
•Especially support medium with ionisable groups e.g.
paper, cellulose acetate membranes etc.
•Electroendosmosis
ii. Molecular sieving
porous sieve-like nature of agarose and polyacrylamide gels
Large pore size faster migration, less frictional force
Small pore size, slower migration, more frictional forces
generated

5. Factors affecting electrophoretic mobility
e) Ionic strength of buffer
i. High ionic strength of buffer:
oIncrease in ion concentration of buffer
Current carried by buffer increase
Current carried by sample decrease
µ decreases
ii. Low ionic strength of buffer
oDecrease in ion concentration of buffer
Current carried by buffer decreases
Current carried by sample increases
µ increases
Ionic strength also affects solubility of sample

6. Factors affecting electrophoretic mobility
f) PH of Buffer
The net charge of the sample especially proteins and
amino acids is determined by pH.
If pH˃pI, the protein migrates to the anode
If pH˂pI the protein migrates to the cathode
If pH=pI, no net movement is observed
The pH does not affect nucleic acids and nucleotides

7. Factors affecting electrophoretic mobility
G. Temperature and heat generated
From the equation heat (H) = I
2
Rt
Where
I is current
R resistance of medium
T time of run.
Heat generated will change the viscosity and density of
the medium Hence alter the rate of migration
Heat will increase convection flow of buffer disturbing
zone of separation.

8. Types of electrophoresis

9. 2 Types of electrophoresis
Zone electrophoresis
Moving boundary
electrophoresis
Electrophoresis
carried out in
solution without
supporting medium
Electrophoresis
carried out in
solution with
supporting medium

10. Moving boundary electrophoresis
Mixture of samples A (negative), B and C (all positve) are placed
in a solution ( buffer). When the PD is applied, the various
samples move to their respective electrodes and are separated as
seen in second figure above

11. Zone electrophoresis
An inert polymeric supporting media is used between the
electrodes to separate and analyze the sample. Could be start,
cellulose acetate, starch, agarose or polyacrylamide

12. Types of Supporting media

13. Sheet of Paper-Paper
Support material is paper made of
polysaccharide called cellulose
Soak the sheet of paper in buffer and
layer on the equipment as shown
Connect wigs to the buffer wells
Place the sample as a tiny drop or thin
line on the paper and close lid to avoid
evaporation
Apply the voltage for separation to go
one for a while
Remove paper, dry it and identify the
bands or sports using colour, staining, uv
light, radiography etc

14. Elution of the separated products
For reasons of downstream analysis, components are
eluted from paper by
Soaking of paper in a small volume of elution buffer
Rinsing a triangular piece of paper with elution buffer and
collecting drops from the tip
Electrophoresing the component of interest from the band into
a small volume of elution buffer

15. Cellulose acetate-cellulose acetate
electrophoresis
Improved form of paper chromatography
OH groups on cellulose that reduce efficiency of the method are
acetylated hence provides better resolution than paper
It has two key advantages over paper
It is transparent, facilitating optical detection of zones
It dissolves easily in a variety of solvents facilitating elution
and isolation of separated components

16. Starch gels
Starch is a polymer made of α
glucose subunits
Apparatus for starch gel is as shown
Heat potato starch in buffer till it
turns transparent and pour in a gel
tank
Cut slots to serve as sample wells
Insert porous wicks as electrical
connections between anodic and
cathodic buffer tanks and gel
Apply sample as a slurry
Seal the top of gel with greese or
wax
After separation, cut sections of gel
for staining
Starch polymer conatinnegatively
charged carboxylate groups that can
Hinder migration of proteins by
interractingwith it
Lead to the phenomenon of
electroendosmosis

17. Polyacrylamide gels -PAGE
used in separating proteins, small RNAs fragments and very
small DNA fragments
Prepared by reacting the monomer acrylamide to the crosslinker
bisacrylamide in the presence of catalyst (TEMED), initiator APS
Pore size determined by the concentration of acrylamide and
bisacrylamide
Experimentally PAGE gels are employed as column or slab gel
systems as seen below

18. Agarose gels
Linear polymers of D-galactoseand 3,6-anhydrogalactose
Prepared by boiling agarose in powder in buffer to form a
continuous gel when cooled to about 38 degree centigrade
Pore size of gel is determined by the amount of agarose
powder used
Can be polymerisedas horizontal slab gels or vverticalslab gels
Viscosity of agarose gels highly sensitive to temperature hence
agarose gels are ran submerged in water