Western%20Blotting

1
Western Blotting
The experiment mainly focuses on the steps
involved in transferring the separated samples
from gel to the membrane and detect the desired
protein by using the fluorescent/enzyme tagged
antibodies.

• Related Los Transfer of gel to membrane,
  fluorescent/enzyme tagged antibodies
• gt Prior Viewing- IDD-11. Protein quantification,
  IDD-17. SDS-PAGE
• gt Future Viewing- IDD-25. SDS-PAGE gel
  analysis, IDD-28. In solution digestion, IDD-30.
  Matrix Instrumentation
• Course NameWestern blotting
  
• Level(UG/PG)UG
• Author(s)Dinesh, Vinayak
• Mentor Dr. Sanjeeva Shrivastava

The contents in this ppt are licensed under
Creative Commons Attribution-NonCommercial-ShareAl
ike 2.5 India license
2
Learning objectives
1

• After interacting with this Learning Object, the
  learner will be able to
• Define the electro-blotting principle
• Operate the steps involved in staining process
• Interpret to detect the protein of interest by
  antibody tagging
• Assess the troubleshooting steps involved in the
  experiments.

2
3
4
5
3
Master Layout
1
Reagents for western blotting (Slide 5-17)
2
Semi dry Transfer (Slide 18)
Western blotting (Slide 19-41)
3
Primary antibody treatment (Slide 42-46)
4
Secondary antibody treatment(Slide 47-52)
5
4
Definitions and Keywords
1
1. Western Blotting The technique used to
transfer the proteins in the gel to the blotting
membrane so that presence of desired protein spot
can be detected using enzyme conjugated
antibodies. 2. Skim milk Milk devoid of cream
or fat is called skim milk which is used for
blocking the membrane to prevent the non specific
binding of the antibodies. 3. Primary Antibody
Antibody which is specific for the protein of
interest. Which has the binding sites for protein
of interest and also for secondary antibody. 4.
Secondary antibody Antibody specific for the Fc
region of the primary antibody which is also
tagged with enzymes like alkaline phosphatase,
which gives a color reaction after reacting with
the substrate like NBT/BCIP. 5.
5-Bromo-4-chloro-3-indolyl phosphate (BCIP) is a
chemical compound used in immunoblotting, with
nitro blue tetrazolium  (NBT), for
sensitive colorimetric detection of alkaline
phosphatase.
2
3
4
5
5
Step 2
1
T1Reagents for western blotting
2
3
Audio Narration (if any)?
Description of the action
Show a measuring balance, with display, ON, OFF
and TARE/0 buttons on it. let user ON it, display
reading as 0.000g, let user picks up the paper
from the rack, makes 1/10 of folding on the sides
and places it on the balance. Now the display
reading changes to 0.003g. Instruct user to TARE
the reading. And animate to click the tare
button. Once user clicks it, reading must show
0
When measuing with paper, the weight of the paper
need to be tared from actual reading.
4
5
Reagents are similar to IDD-32. Buffer
preparation for western analysis from slide 5-17.
6
Step 3
1
T1Reagents for western blotting
Tris base
Glycine
2
methanol
Audio Narration
Description of the action
3
Let user pick up glycine, tris base, methanol,
spatula, measuring cylinder from the rack and
keeps it on the table next to balance. Instruct
user to weigh 28.8g of glycine, let user tare the
balance, user should click on the glycine bottle,
uncap it, with help of spatula weigh the required
amount on a paper over the balance. Display a
gradual increase in reading with quantity
addition. if the gram exceeds user should remove
some quantity or if it less add the quantity to
get the exact required amount. After weighing
transfer the quantity to beaker. Now 4.6g
accordingly for tris base.
Prepare transfer buffer containing glycine, Tris
Base and methanol. Buffer provides wet transfer
condition during electro-blotting of proteins
from gel to membrane. This wet transfer is
recommended for large proteins and avoids drying
of the membrane.
4
5
7
Step 3
1
T1Reagents for western blotting
Tris base
Glycine
2
methanol
Audio Narration
Description of the action
3
Now to the beaker, take methanol bottle, open the
cap, take 1000ml measuring cylinder, measure
900ml. Let user remove the excess methanol if
level crosses 900ml mark. Tranfer it to beaker.
Now take the beaker, shake it to make a proper
mix. Animate the powder getting into the
solution. Now tranfer the beaker solution to
1000ml measuring cylinder to makeup the volume to
1000ml by adding methanol.
If your are using nitrocellulose, buffer must be
prepared using methanol. if using PVDF, distilled
water can be used to prepare the buffer. Methanol
treatment is needed at the time of
electroblotting to activate the PVDF.
Now to the beaker, add methanol
4
5
8
Step 4
1
T1Reagents for western blotting
Tween 20
Tris base
Nacl
2
Kcl
Audio Narration
Description of the action
Let user takes out Nacl, Kcl,Tris Base, tween 20
from the rack and keep it next to balance.
Instruct user to weigh 8g of Nacl, 0.2g of Kcl,
3g of tris base and 600ul of tween 20. let user
pick the bottle, uncap it, weigh the required
amount with help of spatula on a paper over the
balance. Display a gradual increase in reading
with quantity addition. if the gram exceeds user
should remove some quantity or if it less add to
get the required amount. After weighing transfer
the quantity to beaker. Now take out 1ml
pippette, set it for 600ul, take out tween20
bottle, uncap it, pipette and transfer 600ul into
the beaker. .
3
Prepare TBST buffer which can be used as destain
solution and also for washing the membrane.
4
5
9
Step 4
1
T1Reagents for western blotting
Tween 20
Tris base
Nacl
2
Kcl
Audio Narration
Description of the action
3
The pH of the TBST buffer need to set to 7.6.
Now to the beaker, take methanol bottle, open the
cap, take 1000ml measuring cylinder, measure
900ml. Let user remove the excess methanol if
level crosses 900ml mark. Transfer it to beaker.
Now take the beaker, shake it to make a proper
mix. Animate the powder getting into the
solution. Now set the pH to 7.6 by using pH
meter.
4
5
10
Step 5
1
T1Reagents for western blotting
2
STD 1
STD 2
3
Audio Narration
Description of the action
Before the pH reading, pH instrument need to be
calibrated with standards. Once with STD 1 at pH
4 and with STD 2 at pH 9.
Display standard pH bottles and pH instrument
placed on a table. Instruct user to caliberate
the instrument. Let user ON the instrument.
Initially for the pH rod is dipped in water, when
user clicks on read button, display must show a
reading 7. now take out the rod rinse it with
deionized-water, let user cleans the rod with
tissue. Now pick the STD 1 , uncap it, dip the
cleaned rod into the solution, user must click
read button with display showing 4. now clean
the rod and repeat the step to note down the
reading for STD 2.

4
5
11
Step 5
1
T1Reagents for western blotting
2
NaOH
HCl
3
Audio Narration
Description of the action
Instruct user to set the pH for TBST pH at 7.6.
Now take the TBST bottle, uncap it, dip the
cleaned pH rod into the solution. User need to
click on read button. Initially display must show
a reading 6. now instruct user to add NaOH to
adjust the pH. Now allow the user to click on
NaOH bottle so that drops of NaOH should be added
with filler, user need to mix the solution with
glass rod, click on read button and the reading
should anywhere near 6.1- 6.3. let user keeps
adding the NaOH drop till the desired pH is
obtained. and later transfer the beaker solution
to 1000ml measuring cylinder to makeup the volume
to 1000ml by adding methanol
Set the pH of TBST buffer to 7.6.
4
5
12
Step 6
1
T1Reagents for western blotting
Tris base
Nacl
2
Mgcl2
Audio Narration
Description of the action
3
Show the bottles labeled as Nacl, Mgcl2,Tris
Base. The user should click on the required
reagent bottle and spatula for weighing. Instruct
user to weigh 0.585g of Nacl and 0.102 g of
Mgcl2 and 1.11g of Tris and, let user pick the
bottle, uncap it, with help of spatula weigh the
required amount on a paper over the balance. if
the gram exceeds he should remove some quantity
or if it low add to get required amount.
Dissolve the weighed amount by adding 100ml of
methanol (instruct user to measure methanol in
the measuring cylinder) and giving a brief spin
to dissolve it.
Prepare Alkaline phosphatase buffer containing
Nacl,Mgcl2 and tris base which helps in
visualization of bands.
4
5
13
Step 7
1
T1Reagents for western blotting
2
NaOH
3
HCl
Audio Narration
Description of the action
Then the beaker containing(labeled as ALP pH
9.8) 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 pH 8 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 in fillers
and the reading should increase like.8.1,8.3 and
then 8.8,9.2,9.3 and 9..8(desired pH). (follow
the instruction like in slide10 11)
Adjust the pH of the alakaline phosphatase buffer
to 9.8
4
5
14
Step 8
1
T1Reagents for western blotting
2
1000
500
250
3
100
The animator should draw graduated measuring
cylinder as shown in slide with graduation
100ml, 250 ml,500ml,1000ml. The user should
click on the appropriate cylinder for usage
4
5
15
Step 9
1
T1Reagents for western blotting
FIXING SOLUTION
2
WATER
3
Prepare fixing solution by measuring 10ml of
methanol, 7 ml glacial acetic acid and make up
the volume to 100ml with deionized water.
4
5
16
Step 10
1
T1Reagents for western blotting
Sodium Acetate
2
Audio Narration
Description of the action
3
Show the bottles labeled as Sodium acetate and
acetonitrile . The user should click on the
required reagent bottle and spatula for weighing.
Instruct user to weigh 8.2g of Sodium acetate
and, let user pick the bottle, uncap it, with
help of spatula weigh the required amount on a
paper over the balance. if the gram exceeds he
should remove some quantity or if it low add to
get required amount. Dissolve the weighed amount
by adding 80ml of water and 20 ml (instruct user
to measure methanol in the measuring cylinder)
and giving a brief spin to dissolve it.
Prepare destaining solution containing sodium
acetate and acetonitrile
4
5
17
Step 11
1
T1Reagents for western blotting
2
NaOH
3
HCl
Audio Narration
Description of the action
Then the beaker containing(labeled as Destaining
solution pH 4) 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 pH 5 in the display and
instruct user to add Hcl. Now allow the user to
click on Hcl so that drops of Hcl should be added
in fillers and the reading should decrease like
4.8,4.6,4.4,4.2,4)
Adjust the pH of the Destaining solution to 4
4
5
18
Step 1
T2Semi dry Transfer
1
Cathode
2
3
Anode
Semi dry transfer is used for the transfer of
protein spots from gel onto the membrane.
Animator should draw a instrument as shown in the
figure. Include the options like start, time and
stop. Label as Semi dry transfer apparatus
4
5
19
Step 12
1
T3western blotting
2
Anode plate of semi dry transfer instrument
3
4
Place the anode side of the semi dry transfer on
the table
Animator should draw a instrument as in fig and
include the sign
5
20
Step 13
1
T3western blotting
2
Blotting papers
3
4
5
21
Step 13
1
T3western blotting
Animator should draw some white sheets in
rectangular form. Instruct the user to take the
transfer buffer and pour it in the box as given
in figure of previous slide. Animate like the
user taking the white sheets and place it in the
buffer. Instruct the user to make movements on
the box like see-saw.
Place the blot papers in the transfer buffer and
allow it to equilibrate by shaking the box.
2
3
4
5
22
Step 14
1
T3western blotting
2
3
Blot paper
4
Transfer plate
5
23
Step 14
1
T3western blotting
Place the equilibrated blot paper on the semi dry
transfer plate
Animate like taking the white papers from the
transfer buffer and place it on the anode plate
of the semi dry transfer as shown in slide 22
2
3
4
5
24
Step 15
1
T3western blotting
2
Poly vinylidene fluoride
3
4
Methanol
5
25
Step 15
1
T3western blotting
2
Poly vinylidene fluoride
3
4
Transfer buffer
5
26
Step 15
1
T3western blotting
Animator should draw a white sheet and in between
the two blue sheets as shown in figure
slide24. Instruct the user to click on it to
get the name as given in slide The animator
should instruct the user to take out the white
sheet from the blue covering sheets using the
forceps as given in slide and put it in the box
containing methanol Run a clock for 1 minute
and animate like the taking the white sheet and
putting it in the box containing transfer buffer
as in slide24-25 and again show a clock running
a minute.
2
Pre-treat the polyvinylidine fluoride membrane in
methanol followed by transfer buffer for a minute
each.
3
4
5
27
Step 16
1
T3western blotting
2
PVDF membrane
3
4
Blotting paper
5
28
Step 16
1
T3western blotting
Animator should instruct the user to take the
PVDF membrane (white sheet) from the transfer
buffer using forceps and place it on the blot
papers as shown in the figure slide 27
Place the PVDF membrane on the blotting paper and
ensure that there is no bubble between the
membrane and the paper.
2
3
4
5
29
Step 17
1
T3western blotting
2
3
Gel
4
5
30
Step 18
1
T3western blotting
2
3
gel
4
5
31
Step 18
1
T3western blotting
2
Place the SDS-PAGE gel on the PVDF membrane and
ensure that there is no air bubbles between
membrane and the gel.
Animate at a glance the IDD-17. SDS-PAGE for
user to feel the gel run. Animate like placing
the gel on the PVDF membrane and instruct the
user to level it.
3
4
5
32
Step 19
1
T3western blotting
2
Blotting papers
3
4
5
33
Step 20
1
T3western blotting
2
3
4
5
34
Step20
1
T3western blotting
Instruct the user to take the transfer buffer and
pour it in the box as given in figure Instruct
and animate like the user taking the white sheets
and place it in the buffer Instruct the user to
move the box like see-saw. Instruct the user to
place the papers on the top of the gel
Place the blot papers in the transfer buffer and
allow it to equilibrate by shaking the
box. Place it on the top of the gel.
2
3
4
5
35
Step 21
1
T3western blotting
2
3
4
5
36
Step 22
1
T3western blotting
2
3
4
5
37
Step 23
1
T3western blotting
2
3
4
5
38
Step 22 23
1
T3western blotting
2
Pour the transfer buffer on the blot paper Place
the cathode plate on the anode plate containing
blotting set up and connect it to the power
supply Set current depends on the area of the
gel0.8 factor.
Animate like the user pouring the drops of
transfer buffer on the top of the blot paper
using filler as shown in the slide no 50 Show a
cathode plate as in figure slide 36 and instruct
the user to place the plate on the blot
paper Animate like connecting to the power
supply as shown in slide37 Show a power pack
with the options set current, time Instruct
the user to set the current as 50.4 A, time 130
hr.
3
4
5
39
Step 24
1
T3western blotting
PVDF
2
Proteins
3
Gel
4
5
40
Step 25
1
T3western blotting
Animate like the blue squares, red circles, green
triangles from the gel is moving downwards to the
membrane Animate like the blue, red, green
show like disappearing in the gel and appearing
on the PVDF membrane as the time progresses.
During the transfer the negatively charged
proteins migrate from the gel to the membrane in
the positive side
2
3
4
5
41
Step 25
1
T3western blotting
Once the transfer is over remove the membrane
using the forceps avoid body contacts.
Animator should instruct the user to switch off
the power supply after 130 hr Instruct the user
to remove the plate at the top, blot papers using
the forceps to remove the membrane.
2
3
4
5
42
Step 26
T4 Primary antibody treatment
1
2
milk
Rocker
tray
Pour the 5 skim milk 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
membrane into the tray containing milk solution
solution. Now animate user control to take the
tray containing membrane 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
3
Place the membrane in the 5 skim milk solution
and keep it on the rocker to block the
non-specific sites in the membrane.
4
5
43
Step 27
1
T4 Primary antibody treatment
P.Antibody
2
diluted
TBST
tray
3
4
Rocker
5
44
Step 27
1
T4 Primary antibody treatment
Dilute the primary antibody with 11000
concentration using TBST and add the antibodies
to the membrane and shake gently for 2 hours.
Animator should draw a tube labeled as primary
antibody and a bottle labeled as TBST. Instruct
the user to take 1ul of the antibody and 999ul of
TBST using the pipette and transfer it to the new
tube(label as diluted). Draw a tray as given
in the slide 43 containing the membrane
Instruct the user to add the antibody from
diluted tube to the tray Instruct the user to
place it in the rocker and switch on, it should
move slowly like see-saw and show a clock running
2 hours
2
3
4
5
45
Step 28
1
T4 Primary antibody treatment
2
TBST
3
Instruct the user to pour some TBST to the tray
and keep it in the shaker and show a clock
running 10 minutes After 10 minutes show like
removing the TBST from the tray and pouring the
fresh one (repeat the step 3 times)
4
Wash the membrane with TBST to remove the unbound
primary antibodies
5
46
Step 29
1
T4 Primary antibody treatment
2
milk
Rocker
tray
Pour the 5 skim milk 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
membrane into the tray containing milk solution
solution. Now animate user control to take the
tray containing membrane 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
3
Place the membrane in the 5 skim milk solution
and keep it on the rocker to block the
non-specific sites in the membrane and to prevent
the background.
4
5
47
Step 30
1
T5 Secondary antibody treatment
S.Antibody
2
diluted
TBST
Alkaline phosphatase buffer
3
Rocker
4
diluted
tray
5
48
Step 30
1
T5 Secondary antibody treatment
Dilute the Secondary antibody conjugated with
alkaline phosphatase with 15000 conc using
Alkaline phosphatase buffer and 5ml of TBST and
add the antibodies to the membrane
Animator should draw a tube labeled as Secondary
antibody and a bottle labeled as TBST
. Instruct the user to take 1ul of the antibody
and 4999ul of ALP and 5ml of TBST using the
pipette and transfer it to the new tube(label as
diluted). Draw a tray as given in the slide 47
containing the membrane Instruct the user to
add the antibody from diluted tube to the
tray Instruct the user to place it in the rocker
and switch on, it should move slowly like see-saw
2
3
4
5
49
Step 31
1
T5 Secondary antibody treatment
2
TBST
3
Instruct the user to pour some TBST to the tray
and keep it in the shaker and show a clock
running 10 minutes After 10 minutes show like
removing the TBST from the tray and pouring the
fresh one (repeat the step 3 times)
4
Wash the membrane with TBST to remove the unbound
Secondary antibodies
5
50
Step 32
1
T5 Secondary antibody treatment
Rocker
2
BCIP
NBT
3
tray
Animate the above figure with audio narration.
4
 BCIP is the alkaline phosphatase substrate,
which is dephosphorylated by the enzyme and then
dimerizes. This dimer reduces nitroblue
tetrazolim (NBT) to form an insoluble dark blue
diformazan precipitate. Alkaline phosphatase is
commonly conjugated to secondary antibodies.
5
51
Step 33
1
T5 Secondary antibody treatment
2
Negative control
Positive control
Image/graphic for the step
3
Samples
Samples
4
5
52
Step 32 33
1
T5 Secondary antibody treatment
Instruct the animator to add NBT and BCIP from
the tube as shown in figure. Show like placing
the rocker and tray in the dark room Show a
clock running 15 minutes Show the membrane as
given in slide 51 and instruct the user to click
on the bands to know what it is . Draw a bottle
labeled as distilled water and the user should
click on it to pour on the tray for washing the
membrane
Add nitro blue tetrazolium (NBT) and BCIP to the
membrane and keep it in the dark with constant
shaking till the color band appears and wash it
with distilled water. In negative control the
bands will not be seen but in positive control
and samples the bands are visible. The confirmed
protein bands are further used for validation by
MALDI-MS analysis. For more information and
continuity please go through the future IDD.
2
3
4
5
53
Slide 19-41
Slide 42-46
Slide 43-52
Slide 5-17
Slide 18
Tab 02
Tab 03
Tab 04
Tab 01
Tab 04
Name of the section/stage
Animation area
Interactivity area
Interaction 1 Slide 36,37,38 Show like the user
placed the top electrode plate without touching
the bottom plate and animate like the current
showing as 0 in the display Instruction Instruc
t the user to remove the plate and place it
properly on the bottom electrode and switching on
the instruments. Show the current in the display
Instructions/ Working area
Credits
54
Questionnaire
APPENDIX 1
Question 1 Western blotting means a)Transfer of
RNA from the gel to the Blotting
membrane b)Transfer of DNA from the gel to the
Blotting membrane C)Transfer of Proteins from the
gel to the Blotting membrane d)Transfer of
Carbohydrates from the gel to the Blotting
membrane Question 2 Skim milk represents a)Cows
milk b)Milk without caesin c)Milk devoid of
Fats d)Milk devoid of lactose
55
Questionnaire
APPENDIX 1
Question 3 Primary antibody has a)Alkaline
phosphatase tagging b)Fluorescent tagging c)No
tagging d) Color tagging Question 4 Secondary
antibody has a)Alkaline phosphatase
tagging b)Fluorescent tagging c)No tagging d)
Color tagging
56
Questionnaire
APPENDIX 1

• Question 5
• Substrate for Alkaline phosphatase
• Secondary anitbody
• Primary anyibody
• BCIP/NBT
• Skim milk

57
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
58
APPENDIX 3
Summary
The experiment stated is used to detect the
protein of interest using the primary antibody
followed by secondary antibody tagged with the
enzymes when the suitable substrates were added
the color produced will be used detect the
protein spot.