Transcript SDS-PAGE V4

Methodology for the SDS-PAGE
Proteins exhibit different molecular weight depending on the
amino acid composition, this property is exploited to separate
proteins using SDS-PAGE. The electrophoresis method used
to separate proteins based on their molecular weight.
 Related LOs: Electrophoresis, Amino acid property
> Prior Viewing – IDD-11. Protein quantification, IDD-14. Isoelectric focusing, IDD16. Equilibration of IPG strips
> Future Viewing – IDD-20. Silver staining, IDD-22. 2D-gel scanning and image
Analysis
 Course Name: SDS-PAGE
 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:
1.
Define the steps involved in the method
2.
Indicate the buffers/reagents used in the method
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3.
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4.
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5.
6.
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Operate the mechanism of protein separation in SDSPAGE
Outline the gradient gels for proper resolution and
separation of the proteins.
Infer the steps involved to perform Batch processor.
Assess the troubleshooting steps involved in the
experiments.
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Master Layout
Reagent preparation (Slide:5-8)
Buffer Preparation (slide:9-11)
Cocktail for gel casting (Slide:12-13)
3
Gel casting (Slide:14-19)
4
5
Sample loading and gel running
(Slide:20-23)
Display a image from each of these steps, with user click.
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Definitions and Keywords
1.Protein: Proteins are the biomolecules, composing of amino acid, which forms the
basic building blocks of the system and performs most of the biological function of the
system.
2.SDS-PAGE: Sodium dodecyl sulphate –Polyacrylamide Gel Electrophoresis is used
for the separation of proteins based the molecular weight of the proteins. The cocktail
consists of acrylamide, bisacrylamide, TEMED, SDS, Ammonium persulphate.
3.Acrylamide and bisacrylamide: used as a crosslinker for polymerization reaction
to forms the gel like polymer.
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4. TEMED (Tetramethylethylenediamine): catalysis free radical formation of the
ammonium persulphate in the cocktail.
5. Ammonium persulphate
: APS helps in the formation of the free radicals that aid in the polymerization reaction
of acrylamide and bis acrylamide.
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6. Stacking gel: has a acidic pH of 6.8 and a low acrylamide concentration of 5.5%.
Under these conditions proteins separation is poor but form thin, sharply defined
bands at the start of resolving gel
7.Resolving gel: has a basic pH of 8.8, and a higher polyacrylamide of 12.5%. As a
protein, concentrated into sharp bands by the stacking gel, travels through the
separating gel, the pores have a sieving effect, allowing smaller proteins to separate
more faster than higher molecular weight proteins.
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Step 1:
T1: Reagent Preparation
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3
Description of the
action
4
5
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”. This action need to be performed
for each reagent weighing.
Audio Narration
(if any)
Clean the surface of the
balance, Tare the weight
of the paper before
weighing.
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2
3
4
5
Step 1:
T1: Reagent Preparation
Tris-HCL
Water
1.5 M
Tris-Hcl
PH 6.8
1.5 M
Tris-Hcl
PH 8.8
Description of the action
Animator should redraw above figure as shown. Instruct
user to weigh Tris-Hcl, user must tare the balance with
paper like in slide:5, with help of spatula weigh the
required amount (3.15g). Show like the user added
excess amount and show user action to remove the
excess. Transfer weighed amount to tris-hcl ph 6.8 bottle,
followed by addition of water. Animate like the user taking
the water and pouring to the 100ml measuring cylinder till
the volume reaches 80ml and adding to the bottle (repeat
the same weighing and add to bottle labeled as Tris-Hcl (
ph 8.8)). Once water is added let user give a brief spin to
dissolve the reagent into the solution.
Audio Narration
Weigh Tris-Hcl according to
experimental design. Give a
brief spin to dissolve powder
completely. Add half the
amount of total volume of
water required.
Video file: Balancing
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Step 1:
T1: Reagent Preparation
2
NaOH
3
4
5
HCl
Description of the action
Then the beaker containing(labeled as “tris-hcl pH
6.8”) has to be taken near pH meter and allow the
user to dip ph rod in the solution. Animate like the
user switching on the pH meter. The meter should
show 5.5 in the display and instruct user to add
NaOH. Now allow the user to click on NaOH so that
drops of NaOH should be added with fillers and the
reading should increase like 5.8,5.9.6.1,6.3 and
then 6.8(desired pH) and show the making the
solution volume to 100ml in measuring cylinder with
water.
Audio Narration
Prepare 6.8 pH tris –
Hcl solution
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Step 1:
T1: Reagent Preparation
2
NaOH
3
4
5
HCl
Description of the action
Follow above steps to prepare Tris-Hcl ph 8.8. To set the
pH allow the user to dip ph rod in the solution. Animate
like the user switching on the pH meter. The meter should
show 5.5 in the display and instruct user to add NaOH.
Now allow the user to click on NaOH so that drops of
NaOH should be added with fillers and the reading should
increase like 5.8,5.9.6.1, 6.3 and then
6.7,6.9,7.4,7.6,7.8,8,8.4,8.6,8.8(desired pH) and show the
making the solution volume to 100ml in measuring
cylinder with water.
Audio Narration
Prepare 8.8 pH tris –Hcl
solution
Video file: pH meter
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Step 2:
T2: Buffer Preparation
2
3
4
5
Description of the
action
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”. This action need to be performed
for each reagent weighing.
Audio Narration
(if any)
Clean the surface of the
balance, Tare the weight
of the paper before
weighing.
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2
Step 2:
T2: SDS running Buffer preparation
Tris base
SDS
Glycine
Water
3
4
5
Description of the action
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 amount on
a paper over the balance. if the gram exceeds user 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.
Audio Narration
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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Step 2:
T2: Buffer preparation
2
Beaker
Magnetic
bead
3
Description of the
action
4
5
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
Audio Narration
(if any)
Prepare 10X SDS running
buffer. As an when the
powder starts dissolving
make up the volume of
solution to 1000ml by
adding water.
Video file: Magnetic stirrer
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Step 3:
T3:Cocktail for gel casting
2
3
4
5
Description of the
action
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”. This action need to be performed
for each reagent weighing.
Audio Narration
(if any)
Clean the surface of the
balance, Tare the weight
of the paper before
weighing.
1
2
Step 3:
T3:Cocktail for gel casting
SDS
APS
Water
3
4
Description of the action
Show the bottles labeled as APS and SDS.The user should
click on the required reagent bottle and spatula for weighing.
Instruct user to weigh SDS, let user pick the bottle, uncap it, with
help of spatula weigh 0.5g on a paper over the balance. if the
gram exceeds user should remove some quantity or if it low add
to get required amount.
Dissolve the weighed amount by adding 5 ml of water, the user
should click on the pipette, set it to 1000ul to take water and add
to the weighed SDS (repeat 5 times for 5000ul) and giving a
brief mixing on vortex.
The same procedure has to be followed for APS preparation.
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Audio Narration
Prepare 10%SDS
and 10% APS which
are used for the gel
casting.
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Step 4:
T4:Gel casting
B
2
A
3
Description of the action
4
5
The figure shown above is gel running unit.
Please redraw the figure. Instruct user to fix the
glass plates, with spacer in-between the plates on
either sides using glue and clips. animate all the
steps w.r.t figure A.
Once plates are ready let user place them in the
running unit. The final image B.
Audio Narration
Clean the glass plates before
using.Glass plates need to be
fixed in a way there must be no
leakage from sides, proper
sealing must be done.
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Step 4:
T4: Gel casting
Acrylamide
mix
2
3
4
5
Tris HCl
Gel solution
10% SDS
10% APS
TEMED
Description of the action
Show the bottle labeled as Bisacrylamide and
Acrylamide mixture, Tris Hcl (pH8.8) , 10% SDS and
10% Ammonium persulfate, TEMED and water. The
user should click on each bottle to measure the
required quantity. when the user clicks on
Bisacrylamide -Acrylamide, using measuring cylinder
user should pour 3ml and 1.5 ml of TrisHcl to the gel
solution beaker, animate with pipette action for user
adding TEMED, APS and water. (set required volumes
on pipette by listening to audio narration)
Now give a brief spin to mix the gel solution properly.
Audio Narration
Prepare 12.5% resolving gel
solution consisting of 3 ml
acrylamide and bisacrylamide
1.5ml 1.5M Tris –Hcl(pH
8.8),10% 60ul SDS and 10%
60ul APS ,2.11ul TEMED and
1.38ml water.
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Step 4:
T4: Gel casting
2
3
Description of the action
Show the running unit, zoom the glass
plates which are fixed. Now pour the
resolving gel solution into the glass
plates.
4
5
ONce
Audio Narration
Pour the solution at one go, avoid air
bubbles. Pour till the solution covers
the 75% or 3/4th of the glass plate
area. Resolving solution helps for
protein separation.
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Step 4:
T4: Gel casting
Acrylamide
mix
2
3
4
5
Tris HCl
Gel solution
10% SDS
10% APS
TEMED
Description of the action
Show the bottle labeled as Bisacrylamide and
Acrylamide mixture, Tris Hcl (pH8.8) , 10% SDS and
10% Ammonium persulfate, TEMED and water. The
user should click on each bottle to measure the
required quantity. when the user clicks on
Bisacrylamide -Acrylamide, using measuring cylinder
user should pour for 1ml and TrisHcl for 1.8 ml.
To the same beaker, animate with pipette action for
user adding TEMED, APS and water. (setting the
readings as in RHS)
Now give a brief spin to mix the solution properly.
Audio Narration
Prepare stacking gel
consisting of 1 ml acrylamide
and bisacrylamide 1.8ml 1.5M
Tris –Hcl(pH 6.8),10% 75ul
SDS and 10% 75ul APS ,4.5ul
TEMED and 1.6ml water. .
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Step 4:
T4: Gel casting
2
3
4
5
Description of the action
show the running unit, zoom the glass
plates which are fixed. Now pour the
stacking gel solution into the glass plates.
The solution must from a top layer aver
the resolving
ONcegel.
Audio Narration
Pour the solution at one go, avoid air
bubbles. Pour till the solution covers the
top portion of the glass plate area.
Stacking gel helps for sample loading.
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Step 4:
T4: Gel casting
Comb
2
Image/graphic for the step
3
Description of the action
4
5
once stacking gel solution is poured place a
comb on top of the solution. Draw the comb
separately as in figure and animate like the
user placing the comb on the top of the gel.
Place the comb and show like the set up
left for 30 minutes
Audio Narration
Place the comb on the top
of the well so that wells
are formed for loading the
proteins
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Step 5:
T5: Sample loading and gel running
Comb
2
Image/graphic for the step
3
4
5
Sample
Description of the action
After 30m, instruct user to remove the comb. Zoom
to show the wells. Let user take out the samples and
standard from freezer, place them on ice for thawing
for 5min. now let user pipette out the 50ul of
standard by taking the pipette and setting to 50ul to
add in well 1 and similarly for samples in other wells.
After sample and standard loaded, let user fill the
running unit with SDS-buffer. Instruct user to cover
the running unit and place the electrode and press
ON button to start the instrument.
Standard
Audio Narration
Samples in rehydration solution
kept in freezer after sample
extraction and quantification
must be loaded into the wells.
Standards acts like a reference
marker. And bromophenol blue
present in rehydration solution
acts like a indicator.
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Step 5:
T5: Sample loading
Comb
2
3
4
5
Description of the action
Animator should draw a separate Power
pack box and draw options like set voltage,
time, start and stop buttons. Let user make
proper connection of anode and cathode.
Animate like the user should click on set to
set the voltage to 100V and time 1.30hr and
press run.
Audio Narration
After loading the sample, start the gel
run by setting the required voltage and
time.
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Step 5:
T5:Sample loading and gel run
2
3
4
5
Description of the action
Once the running unit is ON.
Animate small air bubbles
coming from positive
electrode and reaching the
top. Also animate that the
sample loaded well, blue color
band travelling from top to
bottom. Re-draw the figure.
Audio Narration
Mechanism of protein separation:
Proteins take up negative charge from SDS present in
the buffer, the negatively charged proteins are attracted
towards the positive end in the presence of electric
field. Proteins start to resolve in gel. Larger protein due
to size, move slowly and get separated at the top. while
smaller proteins tend to resolve into the gel and start
getting separated at the bottom.
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Step 5:
T5:Sample loading and gel run
2
3
4
5
Description of the action
Once after the bromophenol
blue color band reaches the to
positive electrode, instruct
user to stop the run. Let user
take out the connections,
electrode, open the lid, take
out the gel. Now the gel plates
are ready for scanning or for
staining.
Audio Narration
Soon after the blue band reaches the bottom f the gel,
the run must be stopped and gel must be taken for
scanning. For more information on gel scanning do
follow the future viewing IDD.
Slide 58
Tab 01
Slide 911
Tab 02
Slide
12-13
Tab 03
Slide
14-19
Tab 04
Slide
20-23
Tab 05
Tab 06
Tab 07
Name of the section/stage
Animation area
In Slide-20: let user pour SDS-buffer in little quantity or in-between the
run if SDS-buffer level goes low and proceeds with the setup.
Instruction: Display on monitor a very low voltage attained with
comparison to set voltage on the power pack and instruct user to
check the setup again to pour sufficient SDS-buffer till the level
making contact with electrode.
In Slide-21: if user place the electrode the other way round and
proceeds the setup.
Interactivity
area
Button 01
Button 02
Button 03
Instruction: Display on monitor “No voltage, error in setup” and
instruct user to check the setup again to place the electrode
properly red colored wire in “+” port and black colored wire in “-”
port.
Instructions/ Working area
Credits
APPENDIX 1
Questionnaire:
Question 1
What is the use of stacking gel?
a)To separate proteins
b)To increase the resolution
c)To separate the contaminants
d)To make the sharp bands of protein for better separation in resolving gel
Answer: d) To make the sharp bands of protein for better separation in resolving gel
Question 2
pH of resolving and stacking gel
a)8.8 and 6.8
b)6.8 and 8.8
c)6 and 8
d) Same pH
Answer:a) a)8.8 and 6.8
APPENDIX 1
Questionnaire:
Question 3
Charge given by the SDS to the protein is
a) Negative
a)Positive
b)No charge
c)Neutral
Answer: Negative
Question 4
Protein moves from
a)Negative terminal to positive
b)Positive to negative terminal
c)Moves based on the charge of the protein
d)Doesn’t move
Answer: Negative terminal to positive
APPENDIX 1
Questionnaire:
What is the use of resolving gel?
a)To separate proteins based on molecular weight
b)To increase the resolution
c)To separate the contaminants
d)To make the sharp bands of protein for better separation in resolving gel
Answer: To separate proteins based on molecular weight
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
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 resolving gel and stacking gel composition determine the good
separation of protein with high resolution.
The molecular weights of the sample proteins can be estimated with
the help of co-migrated standards with known molecular weights.
The electrophoretic mobility of proteins treated with SDS, depends
largely on the molecular weight of the protein.
Efficiency of the experiment lies in sample preparation, gel casting
with appropriate pH and buffer preparation.