Transcript Passive and Active Rehydration

Passive and Active Rehydration
Proteins from the rehydration buffer has to be loaded into the strips, such that the
strips take up the proteins into the gel for protein separation during IEF. The
process can be carried out in presence and absence of current
 Related Los: IPG strips, IEF property
> Prior Viewing – IDD-6. Extraction of serum protein, IDD-11. Protein quantification
> Future Viewing - IDD-13. Cyanine dye labeling, IDD-14. Isoelectric focusing, IDD17. SDS-PAGE
 Course Name: Passive Rehydration
 Level(UG/PG): UG
 Author(s): Dinesh Raghu, Vinayak Pachapur
 Mentor: Dr. Sanjeeva Srivastava
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Learning objectives
After interacting with this learning object, the learner will
be able to:
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4.
Indentify steps involved in the passive and active
rehydration.
Determine the importance of passive & active
rehydration.
Infer the steps involved to perform the experiment.
Assess the troubleshooting steps involved in the
experiments.
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Master Layout
Cleaning and Drying (Slide:5)
2 Preparation of rehydration solution (Slide:6-8)
Sample addition (Slide:9)
Sample loading (Slide:10)
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Overlay of Strips (Slide:11-12)
Application of Cover Fluid (Slide:13)
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Passive rehydration (Slide:14)
Active rehydration (Slide:15-17)
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Animate two process one for passive and other for active, only final step is different.
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Definitions and Keywords
1. Protein : Proteins are the biomolecules, composed of amino acid, forming the building block of the system
and performs most of the biological functions of the system.
2. IPG strips: Immobilized pH Gradient strips consists of immobilines with that helps in maintaining the pH
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gradients which is exploited for protein separation.
3. IPG buffer: Buffer consists of immobilines which aid in conductance, maintain the stability of separated
proteins
4. Rehydration buffer: A cocktail of reagents used for sample solubalization and used for sample storage.
a)Dithiothreitol: Constituent of rehydration buffer used to reduce the disulfide bonds in the protein.
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b)CHAPS: is a zwitter-ionic detergent, constituent of rehydration buffer that is used to solubilize the proteins
including membrane proteins.
c) Urea: It is a organic compound in rehydration buffer that is used to denature protein.
d)Bromophenol blue: Used in rehydration as color marker and acid –base indicator
5. Cover fluid: Cover fluid prevent evaporation of the sample.
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6. IPGphor IEF instrument: The instrument used for the first dimension separation. It consists of electrodes
with positive and negative terminal with the protocol setting options that can aid in user friendly usage.
7. Passive rehydration: the process of letting the IPG strip to swell with protein sample kept at longer time
without applying current.
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8. Active rehydration: the process of letting the IPG strip to swell with protein sample kept at defined time with
applying current.
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Step 1:
T1:Cleaning and Drying
Reswelling tray
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manifold electrode unit
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Description of the
action/
interactivity
Draw a reswelling tray, manifold unit, a hand
picture, tissue paper. Instruct to click on hand,
place it on the tray to make a movements for
cleaning. Movements should happen when the
user clicks on hand. Draw tray image with water
droplets, later after cleaning with tissue it should
be dry and clean. Please re-draw the figure from
wet to final dry image. Please repeat the same
steps on manifold electrode unit for active
rehydration.
Audio Narration
(if any)
Place the tray/manifold on the
table. Clean all the lanes of
tray/manifold with tissue paper, to
make it free from dust and dry it
completely before use.
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Step 2:
T2: Preparation of rehydration solution
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Description of the
action
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Show a measuring balance. A hand action when
clicked by user should ON the Instrument, pick paper
from rack, place it on the balance so that balance
reads 0.03g and the user should press ”0” on the
balance to make the reading to “0.00”.
Let user tare the balance with paper, before weighing
the reagent.
Audio Narration
(if any)
Clean the surface of the
balance, Tare the weight
of the paper before
weighing.
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Step 2:
T2: Preparation of rehydration Solution
Description of the action
The animator should draw bottles labeled as CHAPS,
urea, dithiothreitol, bromophenol blue, distilled water,
a empty rehydration solution labelled eppendroff tube
and a spoon. Instruct user to click on CHAPS bottle,
unscrew it and take out the powder with help of spoon
to weigh 0.02g followed by urea bottle (0.48g),
dithiothreitol (0.62g), let user takes the pipette set it to
25ul to add bromophenol blue and 1000ul (microlitre)
to add distilled water to the rehydration solution bottle.
Color of solution should change after addition of
bromophenol blue from colorless to blue color. For
more information on preparation refer to slide:8 of
IDD-2. Extraction of plant protein.
Audio Narration
Weigh 8M urea, 2%CHAPS,
40mM Dithiothreitol and add
1%bromophenol blue followed
by 1ml of distilled water.
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Step 3:
T2: Preparation of rehydration solution
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Image/graphic for the step
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Description of the
action
Draw a vortex as shown in figure, user must position
rehydration solution tube on rubber pad of vortex as
shown in figure. Hand action click on the “start”
button and speed knob should regulate the speed of
vortex manually.
Audio Narration
Vortex the tube till, the
solution mixes well for even
distribution.
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Step 3:
T3:Sample addition
Description of the action
The animator should show sample tube, rehydration
solution tube, IPG buffer and an empty tube. Instruct user
to set the pipette to 5ul and add 5ul IPG buffer to empty
tube, set pipette to 300ul to take sample to pipette out into
empty tube. Later set the pipette to 200ul and draw 200ul
of rehydration buffer to add to make total volume 500 ul.
Animate to keep on increasing the level in empty tube
after each addition, now tube is ready for brief vortex.
During vortex make color change of solution in empty tube
to blue color that of rehydration solution. Vortex as shown
in previous slide
Audio Narration
Prepare the sample for
loading it in the strip
equivalent to 600ug
protein concentration for
18cm strip. In case user
is loading for 7cms and
24cms, user can
calculate accordingly
depending upon sample
availability.
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Step 4:
T4: Sample Loading
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Passive rehydration
Active rehydration
Description of the action/ interactivity
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After the vortex, take out and zoom the tube and
instruct the user to draw the solution from tube with
pipette and spread it evenly in single lane of reswelling
tray/manifold. Please redraw the figure with animation.
The user should click and drag the pipette to put the
sample in the lane.
Audio Narration
(if any)
Load the sample in the
reswelling tray/maifold in one
shot, avoid air bubbles. Even
distribution of the sample
must be there and care must
be taken the length of
distribution must be match
length of the strip.
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Step 5:
T5:Overlay of Strips
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Description of the action
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Audio Narration
The animator should draw a freezer named as Remove the strip from -20’C an
20’C and a box inside that labeled as IPG strips
allow it to thaw for 5minutes, before
18cm (4-7 pI). The user should open the door and laying over the sample.
take out the strip pack from the box as shown in
figure. And animate like keeping the strips in room
temperature for 5minutes. Redraw the figure
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Step 6:
T5:Overlay of Strips
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Passive rehydration
Active rehydration
Description of the action
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User must take out the packing cover, with the help of
forceps the strip must be taken out from the packing. Take
out the strip with forceps and in other hand with the help of
another forcep remove plastic cover from the top of the strip
as the user takes it out. Zoom to the strip to show the gel
coating and keep the strip on the top of the sample by
keeping gel side down to contact with sample. The strip must
be laid with the side where the cover was struck, to show gel
side down and lay over the sample solution in the lane at one
go. Show clock for 30min.
Audio Narration
Place the strip on the
reswelling tray in contact
with the sample with the
gel side facing down and
keep it for half an hour
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Step 7:
T6: Application of Cover Fluid
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Passive rehydration
Description of the
action/
interactivity
After 30-60minutes, show a tube labeled as
cover fluid and the user should click on
pipette to take out 1ml cover fluid and
pipette out along the strip as the user click
and drag the pipette as shown in figure.
Active rehydration
Audio Narration
(if any)
Add cover fluid to the strip to
prevent evaporation. While
adding avoid air bubbles. The
strip must be covered with oil.
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Step 8:
T7: Passive Rehydration
Description of the action
The animator should show a clock
running for 16hrs for overnight
rehydration.
Audio Narration
Overnight process helps the IPG strip
to get rehydrated with protein sample
in the absence of electric field. Once
the passive rehydaration is over, the
strip is ready for IEF run. Please refer
the IDD of IEF.
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Step 9:
T7: Active Rehydration
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single electrode manifold unit
Description of the action/
interactivity
For active rehydration. Sample
loading and taking out IPG strip like
in slide: 10, 11, 12 and 13. instead of
using the re-swelling tray user must
use manifold for active rehydration.
cover the electrode with plastic plate
soon after pouring the cover strip oil.
Now transfer the assembly to IEF
instrument.
Strip gel side facing down
Cover Strip oil
Audio Narration
(if any)
Only difference
in active
rehydration
In active rehydration,
sample
is loaded
in a
is
single electrode manifold unit in case of
single strip or strip loaded into manifold
directly if many strips are there. Unlike using
of rehydration tray like in passive rehydration,
for active rehydration manifold is used. The
amount of cover strip oil laid is saved. The
wicks are also saved.
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Step 9:
T7: Active Rehydration
Instead of manifold
tray on the instrument
please re-draw the
figure to keep single
manifold electrode
unit.
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HV ON
light
Description of the action/
interactivity
Let user takes the electrode assembly and
keeps it on the instrument. Place the plastic
support over the electrode assembly. Let
user close the lid of the IPG phor and ON
the instrument. The instrument goes in for
calibration check, animate this on display
and after calibration glow the HV ON light.
Audio Narration
(if any)
The electrode assembly are held
together and placed at the correct
position by the plastic support. The
system goes in for automatic
calibration for display, buzzer and
light to check for working condition.
Step 9: T7: Active Rehydration
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Description of the action/
interactivity
Show the instrument connected with monitor
with desktop display. The user should click on
icon “IPGphor” the window should appear as
shown. Let user have control over selecting the
protocol, number of strip, pH range, set the
voltage and run time for the step-1. let user
connect the instrument and transfer the protocol
to the IPG phor instrument by clicking on Play.
Audio Narration
(if any)
Connect the instrument properly. ON
the system and Select active
rehydration protocol for 5hrs at
20V/50mA for each strip. Now after
the active rehydration step user can
include 1D focusing parameters also.
Do check future viewing IDD for more
information.
Slide 5
Tab01
Slide 68
Tab 02
Slide 9
Tab 03
Slide 10
Slide
11-12
Tab 04
Tab 05
Slide 13
Tab 06
Slide
14
Tab 07
Slide
15-17
Tab 08
Name of the section/stage: 16, 17, 18
Animation area
In Slide-16: if user place the wicks 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 wicks properly
on the gel.
In Slide-17: 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.
Interactivity
area
Button 01
Button 02
Button 03
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.
In Slide-18: if user place the electrode away from the strip and not exactly on the
wicks 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
properly on the wick and the wick making contact with the gel end.
Instructions/ Working area
Credits
APPENDIX 1
Questionnaire:
1)Passive rehydration denotes
a)Movement of protein from the rehydration buffer to the IPG strip Gel with
help of electric field
b)Movement of protein from the rehydration buffer to the IPG strip Gel
without electric field
c)Movement of protein from one IPG Strip to the other with help of electric
field
d)Time to denature protein in the strip
Answer:Movement of protein from the rehydration buffer to the IPG
strip Gel without electric field
APPENDIX 1
Questionnaire:
2)Purpose of IPG buffer
a)Prevent protein renaturation
b)Prevent protein denaturation
c) Maintains conductance during separation
d)All the above
Answer: c)Maintains conductance during separation
3)IPG strips means
a)Immobilized pH gradient
b)Ion-Protein gradient
c)Ionized protein gel
d)Ion-protein gel
Answer: a)Immobilized pH gradient
APPENDIX 1
Questionnaire:
Question 4
IPG strips are used for
a)Protein stability
b)Protein separation based on pI
c)Protein separation based on Molecular weight
d)Protein separation based on amino acid composition
Answer:b)Protein separation based on pI
APPENDIX 1
Questionnaire:
Question 5
Cover fluid prevents
a)Protein denaturation
b) Evaporation of the sample
c)Conductance
d)Gel from melting during separation
Answer:b) Evaporation of the sample
APPENDIX 2
Links for further reading
Reference websites:
2DE Tutorials by Angelika Görg : http://www.wzw.tum.de/blm/deg/
Books:
GE Handbook 2D-Electrophoresis: principle and methods
Biochemistry by Stryer et al., 5th edition
Biochemistry by A.L.Lehninger et al., 3rd edition
Biochemistry by Voet & Voet, 3rd edition
Research papers:
Chen JH, Chang YW, Yao CW et al. Plasma proteome of severe acute respiratory syndrome analyzed by
two-dimensional gel electrophoresis and mass spectrometry.Proc Natl Acad Sci U S A2004,
7;101(49):17039-44.
Eymann C, Dreisbach A, Albrecht D. A comprehensive proteome map of growing Bacillus subtilis cells.
Maldonado AM, Echevarría-Zomeño S, Jean-Baptiste S. et al. Evaluation of three different protocols of
protein extraction for Arabidopsis thaliana leaf proteome analysis by two-dimensional electrophoresis.
Proteomics 2008, 71(4):461-72.
APPENDIX 3
Summary
The experiment involves the sample preparation, loading the sample in strip
and overnight incubation. Enough time must be given for the protein to diffuse
into the strips and occupy the gel in the strips. The strips will be swollen after
rehydration and looks blue as the rehydration enter the strips along with the
proteins. Care should be taken to prevent the bubble formation between the
strip and the sample. Avoid crystal formation and evaporation by covering the
strip with cover fluid