Western Blotting

Western BlottingThe 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 > Prior Viewing- IDD-11. Protein quantification, IDD-17. SDS-PAGE > Future Viewing- IDD-25. SDS-PAGE gel analysis, IDD-28. In solution digestion, IDD-30. Matrix Instrumentation

Course Name:Western 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-ShareAlike 2.5 India license

Learning objectives• After interacting with this Learning Object, the

learner will be able to:1. Define the electro-blotting principle 2. Operate the steps involved in staining process3. Interpret to detect the protein of interest by

antibody tagging4. Assess the troubleshooting steps involved in the

experiments.

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Master Layout

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1Semi dry Transfer (Slide: 18)

Western blotting (Slide: 19-41)

Primary antibody treatment (Slide: 42-46)

Secondary antibody treatment(Slide: 47-52)

Reagents for western blotting (Slide: 5-17)

Definitions and Keywords

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11. 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.

Step 2:

Audio Narration (if any)Description of the action

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3Show 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”

T1:Reagents for western blotting

When measuing with paper, the weight of the paper need to be tared from actual reading.

Reagents are similar to IDD-32. Buffer preparation for western analysis from slide: 5-17.

Step 3:

Audio Narration Description of the action

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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.

GlycineTrisbase

methanol

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.


Step 3:


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3Glycine

Trisbase

methanol


Now to the beaker, add methanol

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.

Step 4:


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3 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.

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NaclTrisbase

Kcl

Prepare TBST buffer which can be used as destain solution and also for washing the membrane.

Tween 20


Step 4:


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3Nacl

Trisbase

Kcl

Tween 20


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.

The pH of the TBST buffer need to set to 7.6.

Step 5:


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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.

STD 1STD 1STD 2STD 2


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.

Step 5:


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3 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.

NaOHNaOHHClHCl


Step 6:


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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.

NaclTrisbase

Mgcl2

Prepare Alkaline phosphatase buffer containing Nacl,Mgcl2 and tris base which helps in visualization of bands.


Step 7:


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3Then 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 slide:10 & 11)

Adjust the pH of the alakaline phosphatase buffer to 9.8

NaOHNaOHHClHCl


Step 8:


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3The 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

100100

250250

500500

10001000


Step 9:

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3 Audio Narration (if any)Description of the action/ interactivity

Show the bottles labeled as ethanol, glacial acetic acid, measuring cylinder and water. Instruct user to measure the reagents, amount must be displayed, click on bottle, pick each bottle to pour the required amount in measuring cylinder. In case if the level is more, instruct user to remove the extra solution. Accordingly, animate pouring and increase in the level of solution simultaneously in the measuring cylinder. Later transfer the solution into the fixing solution bottle.

Prepare fixing solution by measuring 10ml of methanol, 7 ml glacial acetic acid and make up the volume to 100ml with deionized water.

FIXING SOLUTION

WATER


Step 10:


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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.

Sodium Acetate

Prepare destaining solution containing sodium acetate and acetonitrile


Step 11:


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3Then 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

NaOHNaOHHClHCl


Step 1:


T2:Semi dry Transfer

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3Animator should draw a instrument as shown in the figure.Include the options like start, time and stop. Label as “Semi dry transfer apparatus”

Cathode

Anode

Semi dry transfer is used for the transfer of protein spots from gel onto the membrane.

Step 12:

Audio Narration (if any)

Description of the action/

interactivity

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3Animator should draw a instrument as in fig and include the sign

Place the anode side of the semi dry transfer on the table

Anode plate of semi dry transfer instrument

T3:western blotting

Step 13:

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3Blotting papers

T3:western blotting

Step 13: Audio Narration Description of the action

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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.

T3:western blotting

Step 14:

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T3:western blotting

Blot paper

Transfer plate


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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

Place the equilibrated blot paper on the semi dry transfer plate

T3:western blotting

Step 15:

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3Poly vinylidene fluoride

T3:western blotting

Methanol

Step 15:

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3Poly vinylidene fluoride

T3:western blotting

Transfer buffer


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Animator should draw a white sheet and in between the two blue sheets as shown in figure slide:24.

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 slide:24-25 and again show a clock running a minute.

Pre-treat the polyvinylidine fluoride membrane in methanol followed by transfer buffer for a minute each.

T3:western blotting

Step 16:

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Blotting paper

PVDF membrane

T3:western blotting


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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.

T3:western blotting

Step 17:

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3 Gel

T3:western blotting

Step 18:

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3 gel

T3:western blotting


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3Animate 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.

Place the SDS-PAGE gel on the PVDF membrane and ensure that there is no air bubbles between membrane and the gel.

T3:western blotting

Step 19:

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3Blotting papers

T3:western blotting

Step 20:

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T3:western blotting

Step20: Audio Narration Description of the action

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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.

T3:western blotting

Step 21:

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T3:western blotting

Step 22:

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T3:western blotting

Step 23:

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T3:western blotting

Step 22 &23:


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3Animate 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 slide:37

Show a power pack with the options” set current, time”

Instruct the user to set the current as 50.4 A, time 1:30 hr.

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 gel*0.8 factor.

T3:western blotting

Step 24:

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PVDF

Proteins

T3:western blotting


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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

T3:western blotting


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Animator should instruct the user to switch off the power supply after 1:30 hr

Instruct the user to remove the plate at the top, blot papers using the forceps to remove the membrane.

Once the transfer is over remove the membrane using the forceps avoid body contacts.

T3:western blotting

Step 26:


T4: Primary antibody treatment

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3 tray Rocker

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

milk

Place the membrane in the 5% skim milk solution and keep it on the rocker to block the non-specific sites in the membrane.

Step 27:

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Rocker

P.A

ntibody TBST

diluted



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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

Dilute the primary antibody with 1:1000 concentration using TBST and add the antibodies to the membrane and shake gently for 2 hours.


Step 28:


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3TBST

Instruct the user to pour some TBST to the tray and keep it in the shaker and show a clock running 10 minutesAfter 10 minutes show like removing the TBST from the tray and pouring the fresh one (repeat the step 3 times)

Wash the membrane with TBST to remove the unbound primary antibodies


Step 29:


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3 tray Rocker

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

milk

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.


Step 30:

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tray

Rocker

S.A

ntibody TBST

diluted

diluted

Alkaline phosphatase buffer

T5: Secondary antibody treatment


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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

Dilute the Secondary antibody conjugated with alkaline phosphatase with 1:5000 conc using Alkaline phosphatase buffer and 5ml of TBST and add the antibodies to the membrane


Step 31:


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Instruct the user to pour some TBST to the tray and keep it in the shaker and show a clock running 10 minutesAfter 10 minutes show like removing the TBST from the tray and pouring the fresh one (repeat the step 3 times)

Wash the membrane with TBST to remove the unbound Secondary antibodies


Step 32:

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Rocker

NBT

BC

IP


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.


Animate the above figure with audio narration.

Step 33:

Image/graphic for the step

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Samples Samples

Negative control


Step 32 &33:


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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.


Animation area

Instructions/ Working area

Credits

Name of the section/stage Interactivity

area

Tab 02 Tab 03 Tab 04

Button 01

Button 02

Button 03

Tab 01

Slide 5-17

Slide 18

Slide 19-41

Slide 42-46

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

Instruct 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

Tab 04

Slide 43-52

Questionnaire:Question 1:Western blotting meansa)Transfer of RNA from the gel to the Blotting membraneb)Transfer of DNA from the gel to the Blotting membraneC)Transfer of Proteins from the gel to the Blotting membraned)Transfer of Carbohydrates from the gel to the Blotting membrane

Question 2:Skim milk representsa)Cows milkb)Milk without caesinc)Milk devoid of Fatsd)Milk devoid of lactose

APPENDIX 1

Questionnaire:APPENDIX

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Question 3:Primary antibody hasa)Alkaline phosphatase taggingb)Fluorescent taggingc)No taggingd) Color tagging

Question 4:Secondary antibody has

a)Alkaline phosphatase taggingb)Fluorescent taggingc)No taggingd) Color tagging

Questionnaire:APPENDIX

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Question 5

Substrate for Alkaline phosphatasea)Secondary anitbodyb)Primary anyibodyc)BCIP/NBTd)Skim milk

Links for further readingReference websites:2DE Tutorials by Angelika Görg : http://www.wzw.tum.de/blm/deg/

Books:Biochemistry by Stryer et al., 5th editionBiochemistry by A.L.Lehninger et al., 3rd editionBiochemistry by Voet & Voet, 3rd edition

APPENDIX 2

SummaryAPPENDIX

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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.

Western Blotting

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Transcript of Western Blotting