Isofocusing Chromatography

Isofocusing Chromatography
Prepared by- Shirin Akhter
•Iso-focusing chromatography is a
chromatography technique that separates protein
according to differences in their isoelectric point
•Its a polishing step for partially purified samples.
•Its a high resolution technique and can be used
for analytical separation.
•We can be used this technique as a
complementary technique when the protein is not
resolved by other chromatography technique such
as ion exchange, size.
Relationship between protein PH and net charge
•Overall net charge of the protein changes according to
the PH. The PI of each protein is the PH at which the
protein has zero surface charge.
•When the PH above its isoelectric point, protein will
bind to positively charged medium or anion exchanger.
•When the PH below its PI, protein will bind to
negatively charged medium or cation exchanger.
•In isofocusing technique, proteins with different PIs
can be separated by being passed through the
chromatofocusing column which is packed with a
specifically designed medium. Here the PH gradient is
generated on the column by specifically designed and
matched amphoteric buffers. Proteins elute in order to
their isoelectric point.
Figure- Separating proteins according to their
isoelectric points by chromatofocusing.
Mechanism of separation
•Protein separation according to their different PIs,
the chromatofocusing medium is equilibrated with
start buffer at high PH.
•An elution buffer in the column begins to titrate
the amines on the medium and the proteins.
•After pre-gradient volume of elution buffer has
passed, sample is applied to the column.
•Proteins in the sample that are at PH above their
PI are negatively charged which retained near top
of the column.
•Proteins that are at a PH below their PI begin to
migrate down the column and will not bind until
their PH above their PI.
•When the proteins migrate down the column, PH
of surroundings increases and proteins reaches at
PH above their PI which will bind column again.
•The protein with highest PI elute first and the
protein with lowest PI elute last.
Figure: Proteins with different PIs separates as they
pass through the column.
Media and buffer for this technique
We can use different types of media for this
technique such as•Mono P- for fast separation.
•Mono P 5/200 GL- for highest resolution
•PBE 94- for scaling up from Mono P.
•PBE 118- when PH gradient above PH 9.
Different buffers based on the PH
gradient•Polybuffer 74- for PH gradient between 7
and 4.
•Polybuffer 96- for PH gradient should
begin above PH 7.
•Pharmalyte- PH 8- 10.5.
Drawback for this technique
It is less suitable for the isolation of proteins that
precipitate irreversibly at or near their isoelectric
point because these proteins are likely to
precipitate on the column if they reach a high
enough concentration.
Isoelectric focusing:
This is also a high resolution method for the separation
of proteins, peptide and enzymes.
Principle of this method:
In a pH gradient the sample components migrate
towards the anode or the cathode to the pH values,
where their net charges are zero: their isoelectric points
Focusing effect:
A protein diffuse away from its pI, it would gain a
charge and migrate back.
Estimation of isoelectric point for the protein:
The isoelectric points of the proteins can be
estimated with a calibration curve using marker
Isoelectric focusing (IEF) is mostly run in horizontal gels.
The reason behind to run this method in horizontal gels:
•As IEF separates only according to the charge, the gel matrix
must contain large pore sizes. Such a soft gel can slide down
between vertical glass plates. The gel should preferably be
cast on a film support.
•Isoelectric focusing requires efficient cooling and exact
temperature control . This is optimally achieved on a
horizontal ceramics cooling plate connected to a thermostatic
circulator, or a peltier cooling plate.
•Samples have to be applied on a defined pH location
within the pH gradient in order to avoid aggregation
and precipitation of some proteins. This is only
possible on a horizontal gel with an open surface.
•Sharply focused bands can only be obtained with a
high field strength, high voltages have to be applied.
Only horizontal equipment can meet the necessary
safety precautions.
Sample application:
•The samples are applied on the surface of the
•The mode of sample application is dependent on
the sample composition and the IEF gel type.
Because the gradient is sensitive to high salt and
buffer concentrations, it is very helpful to desalt
the samples before application.
The procedure of an IEF run:
•The running condition should be optimized because it
is very important to prevent precipitation and
aggregation of proteins, and to achieve good
•Temperature control is important, because pIs are
highly dependent on the temperature.
•Prefocusing phase should be performed in order to
establish the gradient.
•Samples have to be loaded on the optimized location
with the optimized mode.
•Sample entry should be performed at low field
strength to prevent aggregation.
•The separation time is a compromise between
letting all proteins reach their pIs and keeping the
gradient drift to a minimum.
•After the run, the proteins are fixed by
immunofixation and stained for visualization.
Although isofocusing is foreign to many process
developers, their fractionation characteristic has
advantage to serious evaluation on a routine basis.
It has limitations and it has a distinct set of
development variables, but there are neither more
limitations nor variables than any other technique.
They are simply different. For bulk separation, it
is just as powerful as salt-gradient ion exchange,
hydrophobic interaction, or size exclusion
Thank You

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