Topic 7

Preparative electrophoresis
Source of protein
Objectives
‰ Principle of electrophoresis
Extraction ‰ Basis of protein separation
‰ Factors affecting protein separation
Separation
‰ Types of preparative electrophoresis
‰ Applications

Purity &
characterization

Useful lab reference: Sargent JR and George SG Methods in zone electrophresis

 What is electrophoresis?

‰ Electrophoresis = movement of ions through a solution under the

influence of an electric field. If protein molecules bear charges, they
can also migrate under the influence of electric field

V
- + - +

‰ Migration is due to
‰ motive force - frictional force - electrostatic force
‰ motive force = field strength (Q) x net charge on ion (N)
 Basis of protein separation

Electrophoretic separation of proteins

‰ Due to ability of proteins to migrate in an electric field
‰ Charge on proteins can be altered by pH condition
‰ Proteins migrate towards the anode (+) or cathode (-) depending on
overall charge, except when the pH = pI value of the proteins
(why?)

Basis of separation
‰ By charge and size of proteins (under non-denaturing conditions)
‰ By size of proteins (under denaturing condition eg SDS)
‰ Why? Recall: migration is due to ….
 Factors affecting migration rate

Migration = motive force - frictional force - electrostatic force

‰ Motive force = field strength (Q) x net charge on ion (N)

‰ Frictional force: Resistance encountered by ions attributed to size

and shapes of ions, or viscosity of solution

So, factors that can influence migration rate include

‰Electric field applied
‰Sample type
‰Buffer type and concentration
‰Support medium
 Factors affecting migration rate: Sample type

‰ Charge - migration rate increases with increase charge on proteins.

This is pH dependant
‰ Size - migration rate decreases with increase in size of proteins due
to frictional and electrostatic forces by external medium
‰ Shapes - fibrous or globular proteins exhibit different migration
rates. Reason: different frictional and electrostatic forces

Implication:
‰ Separation of proteins can be due to charge, size and shape (Quiz:
Under what conditions can you achieve this?)
‰ Separation based on size alone (Quiz: How can this be achieved?)
 Factors affecting migration rate:
Applied electric field

‰ Current (i) = voltage (V) / resistance (R)

In electrophoresis, given an applied voltage, current passed is
dependent on resistance exerted by the medium

‰ Potential gradient (PD) = voltage /distance

= V/l volts/m

‰ Force causing migration on an ion bearing a charge of q coulombs

= Vxq/l newtons
Increase in potential gradient (V) will increase migration
 Factors affecting migration rate: Buffer ions

What conducts the current?

‰ Mainly by buffer ions
‰ Small proportion by sample ions

Total charge per second conveyed to the electrode

= nA α Vq/l coulombs per second, where
n = number of ions per m3
A = area of electrode
α = mobility of an ion of q coulombs

Significance:
Increase voltage, increased charge
Distance migrated is proportional to time
 Factors affecting migration rate: Resistance

‰ Resistance to current flow can be caused by

‰ electrophoretic medium
‰ buffer and buffer concentration

Migration and resistance

Total charge, I = nA α Vq/l and I = V/R
1/R = nA α q/l

Significance
Resistance will increase with length (l), decrease with sectional area
(A). An important consideration when using columns - not too long
or too narrow
Influence of buffer on electrophoretic separation

‰ Buffer composition affects migration rate by interacting

with proteins
‰ Buffer concentration, usual range 0.05 to 0.1M
‰high ionic strength buffer
‰slows rate of protein migration
‰increases in overall current and results in higher
heat production
‰too low ionic strength buffer
‰causes diffusion problem and loss of resolution
 Choice of support medium is important

Some effects seen with different medium

‰ Adsorption can lead to tailing effect

‰ Electro-osmosis (with starch, agar and paper)
‰due to movement of H3O+ in buffer to cathode
‰resulting in solvent flow

‰ Molecular sieving effect as seen in polyacrylamide gel

 Electroendoosmosis

Sample slot

+ -

Counter ions (eg cations) Electroendosmostic flow

associated with the starch
medium can move towards
the cathode, resulting in net
movement of water in that
direction
Proteins of isolectric point
equal to buffer pH will flow
towards the cathode
 What happens during electrophoresis

‰ Power dissipated in support medium, W = I2R

‰ Heat generated over a period of time (t) = Wt joules

Possible events
‰ Lowering of resistance due to decrease in viscosity in medium.
This will increase mobility of ions
‰ Heating will cause evaporation of solvent in medium
‰ In turn, buffer ion concentration will increase, resulting in slower
migration of samples (why?)

Prevention
‰ Avoid changes in voltage/current: use constant current or voltage
‰ Cooling system to remove heat generated
 Preparative Electrophoresis

System
‰ Vertical system eg slab or rod/column
‰ Horizontal/ flat bed
Support
‰ Cellulose
‰ Starch
‰ Agarose
‰ Polyacrylamide
Commonly used
‰ Polyacrylamide gel electrophoresis (page)
‰ SDS-page
‰ Isoelectric focusing (IEF)
 Different types of electrophoretic methods

Source: Voet and Voet Biochemistry

 Practical aspects

Columns Horizontal bed

‰ Support is cast in column ‰ Support is cast horizontally
‰ Protein load 100 ng to 50 mg ‰ Protein load easy > 50 mg
‰ More suitable for fast migrating ‰ Suitable for both types of
protein because of the eluting protein species
mechanism
‰ Applications: ‰ Applications:
‰ Page ‰ IEF
‰ SDS-page
‰ IEF
Preparative electrophoresis apparatus

Cathode
chamber (-)

Load sample

Acrylamide gel
(native or SDS)
Separated sample
elution point

Anode
chamber (+)
Separation using SDS-PAGE
 Isoelectric Focusing

Electrophoretic separation of proteins on the basis of

their mobility at a particular pH.
Proteins will migrate to the pH which coincides with its
pI value.
High pH (-)
Sample slot Low pH (+)

pH gradient
 Horizontal bed system

Useful for protein separation by

isoelectric focusing procedure

Source: LKB Multiphor II Manual

 Acetone
fractionation
Using Preparative
Electrophoresis

Source: Boey, CG, Yeoh HH and Chew MY (1976) Phytochemistry 15,

1343-1433
 Purification of tapioca leaf rhodanese
Boey, Yeoh and Chew (1976) Phytochemistry 15, 1343

Leaf Crude
blades extract Supernatant Purity 1.4

Purity 1

Dialysed S/N Purity 1.5

Gel filtration
Preparative Acetone of lyophilised
PAGE fractionation sample
Purity 7.8 Purity 4.3 Purity 2.1
 Purification of tapioca leaf rhodanese

Steps Total Activity Total Protein Specific Purification

umol SCN/15min (mg) Activity Factor
umol
SCN/15min/mg
protein
Crude 1180 979 1.20 1.0
Homogenate
Dialysed 992 545 1.82 1.5
supernatant
Gel Filtration 348 139 2.5 2.1

Acetone 210 41 5.12 4.3

Fractionation
Preparative 150 16 9.4 7.8
PAGE
 Crude enzyme extract

Salt precipitation

Gel filtration Ion exchange

Dialysis
Concentration step

Hydrophobic Electrophoresis Chromatofocusing

Interaction
Chromatography
n itial Affinity
a n i
e d as tep Chromatography
e u s a l s
b in
Can tep or f
s