Methodology for the Equilibration of the strips

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Methodology for the Equilibration of the strips

• Separated proteins in the strip has to
  undergo equilibration step so that the
  multi-subunit proteins can be separated and
  prevention of reunion of the separated proteins,
  stabilization of the separated protein in the gel

• Related LOs IPG strips
• gt Prior Viewing IDD-6. Extraction of serum
  protein, IDD-11. Protein quantification, IDD-14.
  Isoelectric focusing
• gt Future Viewing IDD-17. SDS-PAGE, IDD-20.
  Silver staining,
• Course Name Equilibration
  
• Level(UG/PG) UG
• Author(s) Dinesh Raghu, Vinayak Pachapur
• Mentor Dr. Sanjeeva Srivastava

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Learning objectives
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• After interacting with this learning object, the
  learner will be able to
• Define the preparations prior to the
  second-dimension run.
• Operate the steps involved in handling the
  materials and preparation of buffers.
• Infer the steps involved to perform the
  experiment.
• Assess the troubleshooting steps involved in the
  experiments.

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Definitions and Keywords
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• Equilibration buffer-1 consists of urea as
  denaturing agent, SDS, provide uniform negative
  charge to the protein, DTT , as the reducing
  agent of the disulphide bond, and glycerol as the
  stabilizing agent of polyacrylamide gel in the
  strips.
• Equilibration buffer-II consists of urea as
  denaturing agent, SDS, provide uniform negative
  charge to the protein, IAA , as the acetylating
  agent of reduced disulphide bond, glycerol , as
  the stabilizing agent of polyacrylamide gel in
  the strips.
• Tank buffer consists of Tris base ,glycine, SDS,
  water which aid proper conductance of current
  from one terminal to other that helps proper
  separation of protein.

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Master Layout
1
Preparation of Equilibration buffer-I (Slide5
7)
Preparation of Equilibration buffer-II (Slide6
8)
2
Preparation of Tank Buffer (Slide9-10)
Placing the IPG strip in the equilibration
tray (Slide11)
3
Adding equilibration buffer-I to the strip
(1214)
Mechanism (Slide 13 16)
Adding equilibration buffer-II to the strip
(Slide1517)
4
Washing the strips with tank buffer (Slide18)
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Step 1
T1preparation of Equilibration buffer-I
1

2
Weigh urea, 85 glycerol, pH 8.8 1.5M Tris-HCl
,sodium dodecyl sulphate, 1 bromophenol blue as
per the required volume.
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Step 2
T1preparation of Equilibration buffer-II
1
2
Weigh 10mg/ml of dithiothreitol and 25mg/ml of
iodaoacetamide as per the required volume.
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Step 3
T2preparation of Equilibration buffer-I
1
Urea
1.5 M Tris-Hcl
Sodium Dodecyl Sulphate
2
85 glycerol
1 Bromophenol blue
3
10 mg/ml Dithiothreitol (DTT)
Water
Equilibration buffer-I
The user should click on the tube to get the
constituents of the equilibration buffer I
displayed with audio explanation. Please redraw
the figure
Equilibration buffer-1 consists of urea acts as
denaturing agent, SDS provide uniform negative
charge to the protein, DTT acts as the reducing
agent of disulphide bond, glycerol as the
stabilizing agent of polyacrylamide gel in the
strip.strips
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8
Step 4
T2preparation of Equilibration buffer-II
1
Urea
1.5 M Tris-Hcl
Sodium Dodecyl Sulphate
2
85 glycerol
1 Bromophenol blue
3
25mg/ml Iodoacetamide (IAA) (DTT)
Water
Equilibration buffer-I
The user should click on the tube to get the
constituents of the equilibration buffer II
displayed with audio explanation. Please redraw
the figure
Equilibration buffer-II consists of urea as
denaturing agent, SDS, provide uniform negative
charge to the protein, IAA , as the acetylating
agent of reduced disulphide bond, glycerol , as
the stabilizing agent of polyacrylamide gel in
the strips. the strips
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Step 5
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T2Preparation of SDS-buffer
Tris base
SDS
Glycine
2
Water
Audio Narration
Description of the action
3
Show the bottles labeled as TRIS-Base, Glycine
and SDS. The user should click on the required
reagent bottle and spatula for weighing. Instruct
user to weigh Tris base, let user pick the
bottle, uncap it, with help of spatula weigh the
30.3g on a paper over the balance. if the gram
exceeds he should remove some quantity or if it
low add to get required gram. Follow the same
instruction for weighing 144.1 g of glycine and
10g SDS and transfer all the content into beaker.
Show the measuring cylinder of 1000ml, animate
like the user pouring 500ml of water to the
cylinder and then to the beaker containing
weighed TrisBase, Glycine and SDS and mix it as
shown in slide 15 and instruct the user to pour
it in the measuring cylinder and the reading has
to be 800ml. The user should click on water to
pour 200ml of it in the measuring cylinder
For SDS buffer weigh 30.3g of tris base, 144.1g
of glycine and 10g SDS accurately.
The beaker containing the powder need to be
dissolved into the water.
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Video file Balancing
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Step 6
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T2 Preparation of SDS-buffer
2
Beaker
Magnetic bead
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Description of the action
Audio Narration (if any)?
Prepare 10X SDS running buffer. As an when the
powder starts dissolving make up the volume of
solution in measuring cylinder to 1000ml by
adding water.
Show magnetic stirrer instrument. Let user place
the beaker on it. Display the beaker containing
powder at bottom, liquid layer on top and a
magnetic bead at the bottom. Instruct user to ON
the instrument, let user cotrol the speed nob and
regulate it accordingly to control the mixing
speed in the beaker. Animate powder getting into
the solution. Show a turbid solution turning
colorless Final volume of the solution must be
made to 1000ml in measuring cylinder by adding
distilled water.
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Step 7
T3Placing the IPG strip in the equilibration
tray
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3
Place the IPG strip in the equilibration tray
with gel side up.
Equilibration buffer-I
Animate like the user taking the strip from the
-20C in a tray by opening the freezer taking out
with help of forceps from tray and placing it in
equilibration tray as in figure. For information
please follow the prior IDD. Please redraw the
figures
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Step 8
T4Adding equilibration buffer-I to the strip
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tray
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3
Add equilibration buffer-1 to the strip and keep
on shaker for 15min. The strip must be covered
with the buffer.
Equilibration buffer-I
Zoom equilibration buffer-I tube, and animate
like the user setting the pipette to 1000ul and
pipetting out the buffer when the user clicks on
the pipette (perform two times to get 2000ul)and
show the pipette action along the length of the
strip as in figure and animate like the user
taking the tray and keeping on the rocker and
switching on. Display a clock animation for 15min
along with movement of tray over the shaker.
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Step 9
T4Mechanism of reducing reaction by DTT
1
2
Protein
Show reduced form of bond S-S( put H after each
S)
3
Dithiothreitol in equilibration buffer reduces
the disulphide bond and helps to maintain all
proteins in their fully reduced state.
Equilibration buffer-I
Show the reaction between the dithiothreitol and
disulphide bond of protein. Animate bond
breakage. Please redraw the figure
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Step 10
T4Remove equilibration buffer-I from the lane.
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Remove the strips from the well and place in the
new well
Equilibration buffer-I
After 15min. Animate like the user switching off
the shaker and to stop, and show like taking the
tray in both hand and holding tray by picking
from shaker and using the forceps remove the
strips and put in new well. Please redraw the
figures
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Step 11
T5Adding equilibration buffer-II to the strip
1
2
3
Equilibration buffer-I
Zoom equilibration buffer-II tube, and animate
like the user setting the pipette to 1000ul and
pipetting out the buffer when the user clicks on
the pipette (perform two times to get 2000ul)and
show the pipette action along the length of the
strip as in figure and animate like the user
taking the tray and keeping on the rocker and
switching on. Display a clock animation for 15min
along with movement of tray over the shaker.
Add equilibration buffer-2 to the strip and keep
in shaker for 15min. The strip must be covered
with the buffer. the strips
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Step12
T5Mechanism of acetylation reaction by IAA
1
Protein
2
2 HI
3
Equilibration buffer-I
Animate bond breakage reaction followed by
acetylation reaction. Animate free acetamide
group from IAA going and binding to reduced
disulphide bond. Please redraw the mechanism
IAA acetylates the reduced disulphide bond and
prevent its oxidation, back folding and
aggregating of protein subunits.
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Step13
T5Remove equilibration buffer-II from the
lane.
1
2
3
Remove the strips from the well and place in the
new well
Equilibration buffer-I
After 15min. Animate like the user switching off
the shaker and to stop, and show like taking the
tray in both hand and holding tray by picking
from shaker and using the forceps remove the
strips and put in new well. Please redraw the
figures
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Step14
T6Washing the strips with tank buffer.
1
2
3
Wash the strips with tank buffer after second
equilibration step. Now the strip is ready for
SDS-PAGE run. For more information and continuity
please go through future IDD.
Equilibration buffer-I
Take out the strip from the tray with forceps in
one hand. The user should set the pipette to
1000ul and Take out 1000ul of SDS buffer with
help of pipette. Now pipette out tank buffer
along the strip. Animate the process. Please
redraw the figures
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Slide 1214
Slide 11
Slide 9-10
Slide 1316
Slide 6 8
Slide 5 7
Tab 01
Tab 02
Tab 03
Tab 04
Tab 05
Tab 06
Introduction
Name of the section/stage

• Animation area
• In slide-12 what if user forgets to add
  Equilibration buffer-I and proceeds further.
• Instruction animator must connect the user to
  scanned gel image showing lot of horizontal blue
  lines on the gel.
• In slide-18 after equilibration step and 2D run,
  show a gel image with lines (streaks), spots only
  on ends not in the middle, ask for user input.
• Instruction provide choice like A) uneven
  equilibration (spots only on ends not in the
  middle). B) less concentration of DTT/IAA
  (horizontal lines). user must be able to find
  the right choice.

Interactivity area
Instructions/ Working area
Credits
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Slide 18
Slide 1517
Tab 07
Tab 08
Tab 03
Tab 04
Tab 05
Tab 06
Name of the section/stage

• Animation area

Interactivity area
Instructions/ Working area
Credits
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Questionnaire
APPENDIX 1
Question 1

• The action of DTT in equilibration buffer 1 is
• Oxidation
• Reduction
• Sulfation
• Acetylation
• Answer Reduction
• Question 2
• The action of IAA in equilibration buffer 2 is
• Oxidation
• Reduction
• Sulfation
• Acetylation
• AnswerAcetylation

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Questionnaire
APPENDIX 1

• Question 3
• The reagent glycerol is added for
• Washing the strips
• Stabilizing the gel in the strip
• Separation of the gel from the strip
• Smoothness of the stri
• AnswerStabilizing the gel in the strip
• Question 4
• DTT and IAA acts on
• Electovalent bond
• Covalent bond
• Disulphide bond
• Hydrogen bond

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Questionnaire
APPENDIX 1

• Question 5
• Tank buffer consists of
• Tris base
• Glycine
• SDS
• All the above
• Answer all the above

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Summary
APPENDIX 1
Equilibration of the strips is the important step
that aid in proper separation of the proteins in
second dimension SDS-PAGE. Improper
equilibration will result in streaking and
protein precipitating in the gel forming a
vertical streaking Equilibration solutions has
to be fresh to ensure the proper ingredients are
present which aid in producing good quality gels
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APPENDIX 2
Links for further reading

• Reference websites
• 2DE Tutorials by Angelika Görg
  http//www.wzw.tum.de/blm/deg/
• Books
• Biochemistry by Stryer et al., 5th edition
• Biochemistry by A.L.Lehninger et al., 3rd edition
• Biochemistry by Voet Voet, 3rd edition
• GE Handbook 2D-Electrophoresis principle and
  methods
• 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,
  7101(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.