Off Gel Fractionator
Proteins exhibit unique isoelectric property for a particular
pH, at which the net charge on protein is zero. The
isoelectric property of the proteins are exploited in the
first dimension separation by off gel fractionator
Related Los: proteins isoelectric property, amphoteric molecules, IPG strips,
rehydration buffer
>
Prior Viewing - IDD-11. Protein quantification, IDD-13. Cyanine dye labeling
>
Future Viewing – IDD-16. Equilibration of IPG strips, IDD-17. SDS-PAGE
 Course Name: Isoelectric Focussing/IEF/1D saparation
 Level(UG/PG): UG
 Author(s): Dinesh Raghu, Vinayak Pachapur
 Mentor: Dr.Sanjeeva Srivastava
*The contents in this ppt are licensed under Creative Commons Attribution-NonCommercial-ShareAlike 2.5 India license
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Learning objectives
After interacting with this learning object, the learner will
be able to:
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3.
4.
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5.
Define first dimension separation of proteins using IsoElectric Focusing (IEF) system.
Operate steps involved in handling the instrument and
the materials used.
Recall the background to Iso-Electric Focusing (IEF).
Infer the steps involved to perform the experiment.
Assess the troubleshooting steps involved in the
experiments.
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Master Layout
Place rehydrated IPG strip (Slide:5-9)
Sample loading (Slide:10-11)
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Position Electrode assembly (Slide:12-14)
Switch ON the instrument (Slide:15)
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Set program parameters and run IEF (Slide:16-22)
Mechanism of separation (Slide:23-25)
Collecting of fractions (Slide:26)
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Definitions and Keywords
1. Protein: Proteins are the biomolecules which is composed of amino acid ,forms the
building block of the system and performs most of the function of the system.
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2. IEF: Isoelectric Focusing is an electrophoretic method that separates proteins
according to their isoelectric points (pI).
3. IPG strip: An immobilized pH gradient strip is created by covalently incorporating a
gradient of acidic and basic buffering groups (immobilines) into a polyacrylamide gel at
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the time it is cast.
4. Cover Fluid: Is used to minimize evaporation and thus prevent urea crystallization.
5. Paper Wicks: The wicks absorb excess ions, salts, buffer and aid in conductivity.
6. Electrode assembly: Ensure good contact between the paper wick and IPGStrip gel
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for Iso electric focusing.
7. Manifold: is made of a thermally conductive aluminum oxide ceramic that rapidly
dissipates heat to avoid potential “hot spots.”
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Step 1:
T1:Place rehydrated IPG strip
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3
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Description of the action/ interactivity
Let user takes manifold, a tissue paper
from the rack and keep it on working
table. Let user pick the tissue and
place it on the manifold to make a
movements for cleaning. Animate
manifold image from wet to final dry
image once user is done with cleaning.
Audio Narration
(if any)
Place the manifold on
the table. Take a clean
tissue paper. Clean all
the lanes of manifold,
to make it free from
dust and dry it
completely.
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Step 1:
T1:Place rehydrated IPG strip
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3
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Description of the action/ interactivity
Let user takes IPG strips box from -20'C freezer.
From the box, a pack of strips need to taken out,
let user opens the packing seal, take out the strip
with forceps. Let user open the backing cover
from the strip, and place the strip which has gel
side up in one lane of manifold as shown in the
figure. Let + end of strip be placed on left side of
manifold. Let user makes a note of strip number,
printed on backside of strip.
Audio Narration
(if any)
IPG strips help to
carry out 1D
separation. The strip
has a backing cover,
which need to be
removed before use.
And gel must face up
while carrying out 1D
separation.
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Step 1:
T1:Place rehydrated IPG strip
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3
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Description of the action/ interactivity
Let user takes out the well frame, and places it
on the IPG strip. Let user keeps the right side
of the frame little elevated, place the left side of
the frame against the mechanical stop (about 2
cm, from the end of the tray). Let user apply
little pressure to fix the well frame properly
down until it snaps in.
Audio Narration
(if any)
The well frame have small
compartment, where in sample is
loaded, this helps to hold the
sample on top of strip. Be careful
not to move or crimp the strip while
placing the frame down.
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Step 1:
T1:Place rehydrated IPG strip
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3
4
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Description of the action/ interactivity
Now let user takes out rehydration buffer
tube from the freezer, thaw the tube with
user interaction, pick 200ul pipette, set it
for 40ul and pipette in 40ul of rehydration
buffer in each well. Let user tap the tray
onto the desk, to ensure that the
Rehydration buffer reaches the gel.
Animate clock for 15min and change the
appearance of colorless strip turning into
blue color.
Audio Narration
(if any)
IPG strip need to be swell
with rehydration buffer for
15min. Once the
rehydration buffer has
wetted the gel surfaces
place the wicks.
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Step 1:
T1:Place rehydrated IPG strip
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3
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5
Description of the action/ interactivity
From the kit let user takes out labelled
Electrode Pads, remove four pads for
each IPG strip. Using forceps, wet one
electrode pad by pipetting 0.5ml of
rehydration buffer using 1ml pipette set
for 0.5ml. Place one wetted electrode
pad on each end of the IPG strip. Place
a second wetted electrode pad on top of
the first electrode pad.
Audio Narration
(if any)
User should make sure that
there is no gap between the
pad and the well frame. The
upper pads should be
replaced every 24 hours
of fractionation.
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Step 2:
T2: Sample loading
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3
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Description of the action/ interactivity
Let user takes out the sample solution from the
freezer. Thaw the tube with user interaction,
once the sample turns to liquid form instruct
user to load the sample in wells. Let user pick
200ul pipette, set it for 150ul and pipette in
150ul of sample in each well. Each time
sample is added check for the leak if any
between the wells. If so remove the well frame
and apply pressure and fix it properly with user
interaction.
Audio Narration
(if any)
Sample solution is mixture of
sample and Protein OFFGEL Stock
Solution. Add 150ul of sample in
each well. Check for any leaks
outside the wells. Sample must be
retained into the wells.
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Step 2:
T2: Sample loading
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3
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Description of the action/ interactivity
Let user place the cover seal over the well
frame and press down gently on each well to
secure proper fit. The cover seals have proper
groves to get fit into the wells tightly.
Audio Narration
(if any)
Seal cover avoids evaporation,
be careful not to move the frame
in the tray while applying cover
seal.
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Step 3:
T3: Position Electrode assembly
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3
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Description of the action/ interactivity
Let user reapply 10μL of dH20 onto the
electrode pads at each of the IPG gel ends. Be
careful not to move the pads.
Audio Narration
(if any)
To be on safer side to avoid wicks
from drying add 10μL of dH20.
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Step 3:
T3: Position Electrode assembly
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3
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Description of the action/ interactivity
Instruct user to pipette cover fluid (mineral oil)
onto the gel strip ends. Let user pick 200μl of
pipette to pipette in 200μL cover fluid onto the
anode end (fixed electrode) of the IPG strip.
Similarly pipette 400μL cover fluid at the
cathode side (movable Electrode). Let user
place the tray on the instrument platform.
Audio Narration
(if any)
When pipetting cover fluid,
use caution not to move the electrode
pads. The cover fluid should not
extend
higher than 1/2 the height of the tray
grooves
T3: Position Electrode assembly
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Step 3:
2
Anode electrode
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4
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Description of the action/ interactivity
Instruct user to place electrode. Let user pick
anode electrode (fixed electrode) to place into
the slots on the left side of the tray. Now let user
pick cathode electrode(movable) and place into
the slots on the right side of the tray.
Cathode electrode
Audio Narration
(if any)
The movable electrode should touch the
end of the frame. Be sure that the
moveable electrode makes
contact with the electrode pad. If not,
gently reposition the electrode pad
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Step 4:
T4: Switch ON the instrument
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3
4
5
Description of the action/ interactivity
Let user close the lid of the instrument and
ON the instrument and set the focusing
protocol in the software.
Audio Narration
(if any)
The focusing protocol is the main part
of the experiment.
Step 5: T5:
Set program parameters and run IEF
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2
3
Description of the action/
interactivity
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1 Method selection
2 Start/Stop button
3 Tray corona
4 Status indicator
5 Method step
6 Instrument lock
7 Run parameters
8 Actual values
9 Actuals per lane
Audio Narration
(if any)
Text to be displayed
(if any)
(DT)
Step 5:
T5: Set program parameters and run IEF
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2
3
Description of the action/
interactivity
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5
Redraw the software display like in
previous slide. Let the display
reacts for the user interaction.
Instruct user to edit the status, a
new window like above must pop
up for user to feed in the values.
And let user click on apply to save
the setting.
Audio Narration
(if any)
The parameter setting are for the
focusing step. Depending upon the
strip length used and user
requirement, the parameters can
be changed.
Step 5:
T5: Set program parameters and run IEF
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1 Method selection
2 Parameter per step
3 Timtable graphic
4 Parameter in Timetable
5 Addition of Timetable lines
6 Save/Delete options
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3
Description of the action/
interactivity
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5
Audio Narration
(if any)
Step 5:
T5: Set program parameters and run IEF
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2
3
Description of the action/
interactivity
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5
Redraw the software display like in
previous slide. Let the display
reacts for the user interaction.
Instruct user to add parameters for
HOLD step. And let user click on
apply to save the setting for method
selection. And click on run button to
start the off gel fractionator.
Audio Narration
(if any)
The parameter setting are for
the hold step. The timeline
displays the set voltage, current
and volthour with respect to
time.
Step 5:
T5: Set program parameters and run IEF
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2
3
Description of the action/
interactivity
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5
Display like above soon after the
run button is clicked. Once run
starts, the animator must plot
display the volt attaining as the
time passes.
Audio Narration
(if any)
The graph indicates the run status.
If any problem during the focusing
the volt will not attain the set
voltage, which will be displayed in
the graph plot.
Step 5:
T5: Set program parameters and run IEF
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2
3
Description of the action/
interactivity
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5
If the set voltage is not achieved.
Animator should display it on graph.
Instruct user to stop the run. zoom to
show wick turned to yellow color.
Instruct user to replace it with fresh
one. Once user replace it with fresh
one, instruct him to click on run
button.
Audio Narration
(if any)
In case if there is problem in
run, replace the top wick with
fresh one and start the run
again.
Step 5:
T5: Set program parameters and run IEF
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2
3
Description of the action/
interactivity
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5
If the set voltage is not achieved.
Animator should display it on graph.
Instruct user to stop the run. Display
error “electrode not connected”. Let
user fix the electrode again and fit it
properly. Once done instruct user to
click on RUN button.
Audio Narration
(if any)
In case if there is
problem in run, check
for the electrode
position. Reconnect
the electrode properly
and run again.
Step 6:
T6:Mechanism of separation
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2
3
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Description of the
action/
interactivity
The animator should draw the bands with different
color lines for proteins close to each other. After
applying the voltage show the bands slowly moving
down the strip as the protein separates. Bands
should stop at one place when the sum of charges
is zero. Animation must be done for band movement
with net charge loss/gain.
Audio narration
Protein stops moving at a
point when the net charge is
zero which is called as
Isoelectric point
Step 6:
T6:Mechanism of separation
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2
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Description of the action/
interactivity
Animation must be for band movement
and net charge loss. For net positive
charge the band must loose + ions and
for net negative charge the band must
loose - ion to become zero net charge
after separation.
Audio narration
Protein stops moving at a
point when the net charge is
zero which is called as
Isoelectric point
Step 6:
T6:Mechanism of separation
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2
3
Description of the action/
interactivity
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5
In case of OFF gel Fractionator, animate
the above steps. Red and blue colored
circles are two different types of proteins.
During high voltage animate the circles
must start moving into the well through
the gel as shown above. Once the
focusing is done all red circles must be in
one well and blue ones in other well.
Audio Narration
(if any)
In case if there is problem in run,
replace the top wick with fresh
one and start the run again.
Step 7:
T7:Collecting of fractions
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2
3
Description of the action/
interactivity
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5
Once the run is over, instruct user to stop
the run, and let user open the lid, seal
cover and pick the pipette to take out the
sample from each well and transfer to
fresh tube and label accordingly.
Audio Narration
(if any)
The fraction collected after the run can
be taken for further analysis for more
information please go through through
future viewing IDD.
Slide
1-4
Introduction
Slide 59
Tab 01
Slide
10-11
Tab 02
Slide
12-14
Tab 03
Slide
15
Tab 04
Slide
16-22
Tab 05
Slide
23-25
Tab 06
Slide 26
Tab 07
Name of the section/stage
Interactivity
area
Animation area
INTERACTION 1: In Slide-9: if user place the electrode pad on the strip
backing support rather then on the gel and proceeds the setup.
Instruction: Display on monitor a very low voltage attained with
comparison to set voltage and instruct user to check the setup again
to place the electrode pad properly on the gel.
Button 01
Button 02
Button 03
INTERACTION 2: In Slide-13: while applying the cover fluid, in a hurry or
to test the equipment, let user pour the fluid to a lesser quantity and
proceeds with the setup.
Instruction: Display on monitor a very low voltage attained with
comparison to set voltage and instruct user to check the setup again
to pour sufficient fluid to cover the strip and the fluid level making
contact with electrode.
Instructions/ Working area
Credits
APPENDIX 1
Questionnaire:
Question 1
For a 24cm strip 24 well, what is the total protein load (offgel stock
solution+sample)?
a) 2400ul
b) 3600ul
c) 2424ul
d) 3000ul
Answer: b) 3600ul
Question 2
For a 12cm strip 12 well, what is the total protein load (offgel
stock solution+sample)?
a) 2000ul
b) 1800ul
c) 1212ul
d) 3000ul
Answer: b) 1800ul
APPENDIX 1
Questionnaire:
Question 3
The Glycerol is used for
1) Reduces electroendosmotic effects
2) Prevents drying of gel
3) Prevents urea crystallization
a) I Only
b) II Only
c) III only
d) I, II and III
Answer: d) I, II and III
Question 4
Bromophenol blue allows?
a) Control of liquid distribution
b) To color the protein
c) To remove the protein charge
d) To add charge on the protein
Answer: a) Control of liquid distribution
APPENDIX 1
Questionnaire:
Question 5
Charge on the protein at lower pH range is
a) Positive charge respectively
b) No charge and Negative charge respectively
c) Negative charge and no charge respectively
d) Positive charge and no charge respectively
Answer: a) Positive charge respectively
APPENDIX 2
Links for further reading
 Reference websites:
GE Handbook 2D-Electrophoresis: principle and
methods
Research papers:
1.http://www.chem.agilent.com/Library/brochure
s/5990-5596EN.pdf
2.https://intramural.nhlbi.nih.gov/Cores/PAF/Doc
uments/Instruction%20for%20OFFGel.pdf
3. Jerome Chenau et, al., Peptides OFFGEL
electrophoresis: a suitable pre-analytical step
for complex eukaryotic samples fractionation
compatible with quantitative iTRAQ labeling.
Proteome Science 2008, 6:9
APPENDIX 3
Summary
The experiment mostly focuses on the first dimension separation of the
proteins based on their isoelectric point. The process differs from
conventional gel electrophoresis, as in that the sample components
separated as fractions can be recovered after electrophoresis. The
experiment will be successful only when the sample is free of impurities and
the run goes without any interference.