Phospho Staining

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Phospho Staining
The experiment mainly focuses on the staining of
proteins which are phosphorylated during the post
translational modification

• Related LOs Staining techniques.
• gt Prior Viewing IDD-14. Isoelectric focusing,
  IDD-17. SDS-PAGE, IDD-18. Second dimension
  separation of proteins, IDD33 Western blot
  assay.
• gt Future Viewing IDD-22. 2D-gel scanning and
  image Analysis, IDD-26. Spot picking
• Course Name Methodology for Phospho staining
• 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-ShareAl
ike 2.5 India license
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Learning objectives
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• After interacting with this learning object, the
  learner will be able to
• Define the preparation steps for staining
• Identify the mechanism behind the staining
• Operate the steps involved in getting good
  proteome profile
• Infer the steps involved to perform the
  experiment
• Assess the troubleshooting steps involved in the
  experiments.

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Master Layout
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Definitions and Keywords
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• Pro-Q diamond stain fluorescent dye that stains
  the phosphorous group in the protein that can be
  easily seen when the gel/membrane exposed to the
  UV light/LASER.
• Fixing solution consists of ethanol, glacial
  acetic acid and water. The solution helps to
  precipitate the protein in the gel.

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Step 1
T1 Fixation
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Once proteins are separated on SDS-PAGE, they
need to be transferred onto PVDF membrane by
Electroblotting process. After electro-blotting
of proteins on to the membrane, which later need
to be dried to carry out staining process.
Animator explain the process with the separation
of sample by SDS-PAGE, transfer of gel proteins
onto a PVDF or nitrocellulose membrane by
standard procedures like explained in IDD33
Western blot assay. Instruct the user to go
through prior viewing IDDs or display the images
from the same at a glance.
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Step 1
T1 fixation
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Prepare the fixing solution and transfer it to
the tray. Fixation step helps to fix the
proteins, remove the excess SDS which may
interfere in the staining process and also modify
the proteins to enhance the staining reaction.
Animate bottles labeled as Fixing solution,
concentrated acetic acid, deionized water (dH2O),
methanol, measuring cylinder MC, let user picks
up these from the rack. Instruct user to prepare
Fixing solution. Add 7mL of concentrated acetic
acid in 10ml MC to 80mL deionized water (dH2O) in
100ml MC, add 10mL of methanol, bring the volume
up to 100mL with dH2O, and mix thoroughly.
Animate the steps with user control.
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Step 2
T1 fixation
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Description of the action
Audio Narration (if any)?
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Animate like the user takes the PVDF membrane
after electroblotting. Animate like user takes
the membrane and put it in the tray containing
fixing solution and show a clock running for 10
minutes
Place the membrane in the fixing solution for 10
minutes on a shaker.
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Step 8
T2 Sensitization
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gel
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Animator should instruct the user to discard the
previous liquid into the waste by tilting the
tray or taking out the cap from the opening at
the bottom of the tray. now place the gel in the
ultra-pure water and allow it incubate 10
minutes Show a clock running 10 minutes
Place the gel in ultrapure water for 10 minutes.
Washing is carried out to remove the fixing
solution. Washing can be done two or three times,
each time with fresh water.
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Step 9
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T2 Sensitization
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Instruct user to prepare Reagent1. Place the
required materials on the table, like
Sulfosalicylic acid bottle, acid, water and
measuring cylinder. Let user measure 10ml of
Sulfosalicylic acid, transfer it to beaker, now
measure 90ml of water in 100ml MC and add to
beaker. Let user pick the beaker and shake it to
mix the solution.
Reagent 1 helps to remove unwanted free and low
molecular weight phospho compounds if any from
the PVDF membrane.
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Step 9
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T2 Sensitization
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Transfer the gel to the tray containing
sulfosalicylic acid and allow it incubate for 15
minutes.
Animator should draw a bottle labeled as reagent
1 Sulfo salicylic acid. Animator should instruct
the user to discard the previous liquid into the
waste by tilting the tray or taking out the cap
from the opening at the bottom of the tray.
Instruct the user to pour the reagent1 to the
tray containing the gel and leave it for 15
minutes Show a clock running 15 minutes
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Step 10
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T3 Staining
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Animate bottles labelled as Reagent 2,
Sulfosalicylic acid, deionized water (dH2O)
Cacl2 measuring cylinder 100 mL. Instruct user
to prepare Reagent 2. Add 10ml of Sulfosalicylic
acid acid to 80 mL deionized water (dH2O) in
100ml MC add 10 mL of Cacl2 bring the volume up
to 100 mL with dH2O and mix thoroughly. Animate
the steps with user control.
Reagent 2 helps to increase band sharpness.
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Step 10
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T3 Staining
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Transfer the gel to the tray containing
sulfosalicylic acid and calcium chloride and
allow it incubate for 15 minutes
Animator should draw a bottle labeled as reagent
2 Sulfo salicylic acid Calcium
Chloride. Animator should instruct the user to
discard the previous liquid into the waste by
tilting the tray or taking out the cap from the
opening at the bottom of the tray. Instruct the
user to pour the reagent2 to the tray containing
the gel and leave it for 15 minutes. Show a clock
running 15 minutes
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Step 11
T3 Staining
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Animator should draw a bottle labeled as
ultrapure water let user discard the previous
liquid into the waste. Let user pick ultrapure
water, measure around 250ml and pour into the
tray containing the gel and leave it for 5min.
Transfer the gel to the tray containing ultrapure
water for washing. Washing is carried out to
remove residual solution from previous step.
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Step 12
T4 Developing
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Instruct user to prepare Reagent3. Place the
required materials on the table, like weighing
balance, NaOH bottle, water and measuring
cylinder. Let user weigh 20g of NaOH, transfer it
to beaker, now measure 1L of water and add to
beaker. Let user pick the beaker and shake it to
mix the solution.
Reagent3 helps in hydrolysis to free more
phosphate for staining.
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Step 12
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T4 Developing
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Animator should draw a bottle labeled as reagent
3 0.5 N NaOH Instruct the user to discard the
previous reagent, now let user pour the reagent3
into the tray containing the gel and place it in
the incubator for 20 minutes at 65' C. Show a
clock running 20 minutes
Transfer the gel to the tray containing 0.5N NaOH
and place it in incubator for 20 minutes at 65' C.
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Step 13
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T4 Developing
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Instruct user to prepare Reagent4. Place the
required materials on the table, like weighing
balance, ammonium molybdate bottle, water and
measuring cylinder. Let user weigh 1gram of
ammonium molybdate, transfer it to beaker, now
measure 1Litre of water and add to beaker. Let
user pick the beaker and shake it to mix the
solution.
Reagent4 produces molybdate ions which penetrate
the gel and form phospho-molybdate complex.
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Step 13
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T4 Developing
Transfer the gel to the tray containing Ammonium
Molybade solution and allow it incubate for 10
minutes Repeat the step once more.
Animator should draw a bottle labeled as reagent
4 Ammonium Molybate solution Instruct the user
to discard the previous reagent, now let user
pour the reagent4 into the tray containing the
gel and leave it for 10 minutes Show a clock
running 10minutes repeat this step again by
preparing reagent4 once again.
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Step 14
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T4 Developing
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Instruct user to prepare Reagent5. Place the
required materials on the table, like weighing
balance, ammonium molybdate bottle, nitric acid,
water and measuring cylinder. Let user weigh 1g
of ammonium molybdate, transfer it to beaker, add
10ml of nitiric acid to beaker, now measure 1L of
water and add to beaker. Let user pick the beaker
and shake it to mix the solution.
Reagent5 helps to remove free molybdate ions.
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Step 14
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T4 Developing
Transfer the gel to the tray containing Ammonium
Molybdate nitric acid solution and allow it
incubate for 20 minutes
Animator should draw a bottle labeled as reagent
5 Ammonium Molybdate nitric acid
solution Instruct the user to discard the
previous reagent, now let user pour the reagent5
to the tray containing the gel and leave it for
20 minutes Show a clock running 20minutes
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Step 15
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T5 Stopping
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Instruct user to prepare Reagent6. Place the
required materials on the table, like weighing
balance, methyl bottle, acetic acid, water and
measuring cylinder. Let user weigh 5g of amethyl
green, transfer it to beaker, add 10ml of acetic
acid in 25ml MC to beaker, now measure 1L of
water and add to beaker. Let user pick the beaker
and shake it to mix the solution.
Reagent6 helps in staining the phospho-molybdate
complex. The complex formed takes up the dye in
presence of acetic acid.
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Step 15
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T5 Stopping
Transfer the gel to the tray containing methyl
green solution and allow it incubate for 20
minutes. gel takes up the stain and protein spots
are visible.
Take out bottle reagent 6 methyl Green
Solution. Instruct the user to discard the
previous reagent into the waste and let user pour
the reagent6 into the tray containing the gel
and leave it for 20 minutes Show a clock
running 20minutes
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Step 16
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T5 Stopping
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Step 16
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T5 Stopping
Transfer the gel to the tray containing
sulfosalicylic acid and allow it incubate for 15
minutes
Animator should draw a bottle labeled as reagent
1 Sulfo salicylic acid Instruct the user to pour
the previous reagent into the waste and pour
reagent1 to the tray containing the gel and
leave it for 15 minutes Show a clock running 15
minutes Each step must happen when the user
clicks on the hand
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Step 17
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T5 Stopping
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Instruct user to prepare Reagent7. Place the
required materials on the table, like acetic
acid, water and measuring cylinder. Let user
measure 7ml of acetic acid, transfer it to
beaker, now measure 1L of water and add to
beaker. Let user pick the beaker and shake it to
mix the solution.
Reagent7 helps in destaining the gel.
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Step 17
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T5 Stopping
Transfer the gel to the tray containing 7 acetic
acid for de-staining and allow it incubate for 15
hours (overnight)
Animator should draw a bottle labeled as 7
acetic acid Instruct the user to pour the
reagent to the tray containing the gel and leave
it for 15 hours (overnight) Show a clock running
15 hours (overnight)
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Step 18
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T6 Preservation
standard molecular band
Standard Molecular band
Sample band
Standard Molecular band
Sample band
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Step 18
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T6 Preservation
Animator should show the gel as in figure like in
previous slide.
Once the gel image is obtained after staining
user can go through future viewing IDD for more
information.
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Slide 5
Slide 6,7
Slide 8
Slide 9
Slide 10-12
Slide 13
Slide 14,15
Tab 02
Tab 03
Tab 04
Tab 05
Tab 06
Tab 07
Tab 01
Name of the section/stage
Animation area In Slide-26 let user interpret
the result of the bands (arrow marked) with the
bands of standard molecular marker. Instruction
provide some near values for user to select, in
according to the sample bands to that of standard
molecular band.
Interactivity area
Instructions/ Working area
Credits
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Slide 20,21
Slide 16,17
Slide 22,23
Slide 18,19
Slide 24,25
Slide 26,27
Slide 28,29
Tab 09
Tab 10
Tab 11
Tab 12
Tab 13
Tab 14
Tab 08
Name of the section/stage
Animation area
Interactivity area
Instructions/ Working area
Credits
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Slide 34,35
Slide 30,31
Slide 32,33
Tab 16
Tab 17
Tab 15
Name of the section/stage
Animation area
Interactivity area
Instructions/ Working area
Credits
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Questionnaire
APPENDIX 1
Question 1 What is Pro Q diamond? a)Glucostain b)
Phosphostain c)Protein stain d)DNA
stain Question 2 What is reagent1? a)Sulfsalicy
lic acid b)Nitro salicylic acid c)Ammonium
molybdate d)Calcium chloride Question 3 What
is the stain used in gel code stain? a)Methyl
orange b)Methyl red c)Methyl green d)Methylene
blue
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Questionnaire
APPENDIX 1
Question 4 Pro-Q diamond and Methyl green
stains a)Phospho stains b)Phosphorous in the
cell c)Phospho proteins d)Glucoproteins Question
5 Pro-Q diamond stained gel is scanned in
visible light gel scanner at a)555nm b)666nm c)77
7nm d)888nm
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APPENDIX 2
Links for further reading

• Reference websites
• http//www.piercenet.com/browse.cfm?fldID02051029
  
• Research papers
• Molecular probes(2007).Pro-Q diamond
  phospho-protein blot stain kit.

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APPENDIX 3
Summary
The method detect phosphoproteins directly in
1-D and 2-D gels. Steps involved are fixing,
staining and destaining, no blotting or
antibodies required. Detect phosphate groups
attached to tyrosine, serine or threonine
residues. Signal is linear over three orders of
magnitude and correlates with the number of
phosphates present.