Related Los: Dye binding properties

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Methodology for the Commassie staining
Proteins that are separated by the 2-DE has to be
stained by the staining solution for
visualization. Many staining methods are
available for visualization of proteome, choosing
appropriate method to detect low concentration of
protein is essential for the analysis

• Related Los Dye binding properties
• gt Prior Viewing IDD-17. SDS-PAGE, IDD-18.
  Second dimension separation of proteins
• gt Future Viewing IDD-22. 2D-gel scanning and
  image Analysis, IDD-26. Spot picking
• Course Name Commassie staining
  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 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
1
Preparation of Fixing solution (Slide5-6)
2
Fixing of gel (Slide7)
Preparation of staining solution (Slide8)
3
Staining of gel (Slide9-10)
Preparation of destaining solution (Slide11)
4
Destaining of gel (Slide12)
Washing of gel (Slide13-15)
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Definitions and Keywords
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1) Coomassie blue The staining dye in the
staining solution is negatively charged dye that
binds to the protein and makes protein visible as
a spot in the gel. 2) Staining solution The
solution consists of methanol, acetic acid, water
and coomassie blue that stains the proteins. 3)
Destaining solution The solution consists of
methanol, acetic acid and water, which helps to
remove unwanted stains or excess stains from the
gel and helps to retains the stain of proteins.
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Step 1
T1 Preparation of Fixing solution
1
2
1000
500
250
3
100
The animator should draw graduated measuring
cylinder as shown in slide with graduation
100ml, 250ml, 500ml, 1000ml. Let user take out
these cylinder from the rack.
The user should click on the appropriate
cylinder for usage, when needed should clean and
rinse with little amount of distilled water
before use.
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5
6
Step 1
T1 Preparation of Fixing solution
1
FIXING SOLUTION
2
WATER
3
Prepare fixing solution by measuring 75ml of
ethanol, 25 ml glacial acetic acid and make up
the volume to 250ml with water. Fixing solution
helps protein fixation on to the gel and avoid
protein diffusion.
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7
Step 2
T2 Fixation of gel
1
2
GEL
Staining tray
Rocker
3
Place the gel in the fixing solution to remove
the compounds like SDS, ampholytes that produces
background. In the meantime prepare the reagent
for the next step.
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8
Step 3
T3 Preparation of staining solution
1
2
coomassie blue tablets
3
Animator should draw the bottle labeled as
methanol, acetic acid, coomassie blue tablets and
water, let user takes these things from the rack
and place it on the working bench. The user
should click on methanol bottle to measure 500ml
in 1000ml measuring cylinder(MC), and measure
70ml of Acetic acid in 100ml MC, measure 430 ml
of water in 500ml MC pour all the measured
solutions into 1000ml MC to make the volume to
1L. Show the user opens the bottle for commassie
tablet and take a tablet (blue color) and put in
the paper and draw a hammer so that when the user
clicks on the hammer the tablet has to be crushed
to powder and add the fine powder made to the
above made solution. The colorless solution
should turn blue and transfer it to staining
solution Bottle. Animate user keeping the bottle
for magnetic stirrer to dissolve the powder
completely.
Measure the required quantity of acetic acid,
methanol and water and add a coomassie tablet
powder for 1 liter of the reagent.
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9
Step 4
T4 Staining of gel
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2
Rocker
Staining tray
Place the gel in the coomassie staining solution
containing methanol ,acetic acid and coomaassie
blue and keep it in the shaker overnight and
cover it to prevent evaporation. In the meantime
prepare the reagent for the next step.
Pour the staining solution into the tray with
user interaction. Instruct user to stop the
rocker, pick the tray containing gel from the
rocker, place it on table. Now transfer the gel
into the tray containing staining solution. In
other way discard the fixing solution into
discard bottle, by slanting the tray and holding
the gel. In some case, tray will have opening at
the bottom on sides which can be opend to discard
the solution. Now animate user control to take
the tray containing gel to be placed on ROCKER.
Allow user to set the parameters for the rocker
and to start the instrument. Animate see-saw
movement for the rocker along with solution
movement in the tray
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10
Step 4
T4 staining of gel
1
2
protein
Protein-dye complex
Dye
3
The dye molecules bind to proteins to form a
protein-dye complex. The formation of the complex
stabilises the negatively charged anionic form of
the dye producing the blue colour.
4
Coomassie blue binds to protein through ionic
interactions between negatively charged sulfonate
group in dye and amine groups in protein and
Vander wall attractions as well.
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Step 5
T5 Preparation of De-staining solution
1
De-stain solution
2
3
Animator should draw the bottle labeled as
methanol, acetic acid and water. The user should
click on methanol bottle to measure 500ml in
1000ml measuring cylinder, and measure 70ml of
Acetic acid in 100ml measuring cylinder, measure
430 ml of water in 500ml measuring cylinder pour
into 1000ml measuring cylinder to make
De-satining solution. The solution must look
colorless
Measure the required quantity of acetic acid,
methanol and water for preparing destaining
solution which removes unwanted dye stains in the
gel.
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Step 6
T6 De-staining of gel
1
DE-STAINING SOLUTION
GEL
2
Rocker
3
Place the gel in the De-staining solution for
6hours with gentle shaking till the dyes are
removed from the gel. Replenish the solution
several times until background of the gel is
fully destained. Once the protein profile is
visible you can stop the rocker for the next
step.
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Step 7
T7 Washing of gel
1
water
GEL
2
Rocker
3
Place the gel in the water solution for 2hours
with gentle shaking.
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14
Step 7
1
T7 Washing of gel
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3
Gel showing protein sample stained with
coommassie blue. In comparison to other staining,
this method is medium sensitivity, steady state
method, fast, helps for good quantification,
inexpensive, very environment friendly, mass
spectrometry compatible.
The animator should show the gel as in the slide
after the staining is done.
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15
Step 7
1
T7 Washing of gel
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3
Once user have the stained image, user can
proceed to scanning, followed by Gel analysis. In
some case after staining the gel can be used for
spot picking. Please go through following IDD
for more information.
The animator should show the gel as in the slide
after the staining is done.
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16
Slide 9-10
Slide 13-15
Slide 11
Slide 12
Slide 5-6
Slide 8
Slide 7
Tab 02
Tab 03
Tab 04
Tab 05
Tab 06
Tab 07
Tab 01
Name of the section/stage
Animation area In slide 14 Animate on the
stained image some fine blue points/spots and let
user finds out the reason behind it. Instruction
1.fine blue points/spots uneven mixing of
coomassie powder into the solution. Instruct user
to carry out the mixing step again and later
carry out de-staining step. 2.fine blue
lines/spots tissue paper residue on the gel.
Instruct user to use a cleaned staining tray and
carry out de-staining step.
Interactivity area
Instructions/ Working area
Credits
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Questionnaire
APPENDIX 1
Quiz Question 1 The charge of coomassie dye
is a)Negative b)Positive c)Neutral d) Both
ab Answer a)negative Question 2
The coomassie dye stains the protein as a result
of a)Proton transfer b)Hydroxyl ion transfer c)
Electron transfer d) None of the above Answerc)
Electron transfer
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Questionnaire
APPENDIX 1
Question 3 Constituents of staining
solution 1)Methanol 2) Acetic acid 3) water
4)coomassie blue 5)blue dye a)1 2 b) 23 c)all
the above d)1,2,3,4 Answer d)1,2,34 Questio
n 4 Constituents of destaining
solution 1)Methanol 2) Acetic acid 3) water 4)
bromophenol blue 5)blue dye a)1 2 b) 23 c)all
the above d)1,2,3 Answerd)1,2,3
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Questionnaire
APPENDIX 1
Question 5 Commassie dye detects protein in
range of a)Micrograms b)Milligrams c)Nanograms d)
Kilograms Answer a)Micrograms
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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 Bioch
emistry 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, 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.
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APPENDIX 3
Summary
Negatively charged coomassie dye bind to the
protein and make the protein to be visible as a
spot in the gel. Protein spot takes up dye color
once it is stained with Commassie stain.
The staining technique is less sensitive and it
detects protein of microgram concentration. This
staining method is medium sensitivity, steady
state method, fast, good quantification,
inexpensive, very environment friendly, mass
spectrometry compatible.