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Western Blotting Western Blotting

Bangalore Genei, 2007 Bangalore Genei, 2007

GeNeiTM

GeNeiTM

GeNei

TM

Western BlottingTeaching Kit

Manual

Cat No. New Cat No.

KT21 106129

KT21A 106160

KT21B 106161

Revision No.: 00260705

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CONTENTS

Page No.

Objective 3

Principle 3

Kit Description 5

Materials Provided 7

Procedure 11

Observation & Interpretation 14

ORDERING INFORMATION 1 6

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Objectives:

To learn the technique of Western Blotting which involvesthe following experiments:

Electrophoresis of the protein (SDS-PAGE)

Electrotransfer of protein onto nitrocellulose membrane(Western Blotting)

Immunodetection of the transferred protein(Blot development)

Principle:

Western blotting (also known as protein blotting orimmunoblotting) is a rapid and sensitive assay for detectionand characterization of proteins. Western Blotting techniqueexploits the inherent specificity of antigen-antibody interactionto identify specific antigens by polyclonal or monoclonalantibodies.

SDS-PAGE:

Sodium Dodecyl Sulphate - PolyacrylamideGel Electrophoresis (SDS-PAGE ) is carried out in adiscontinuous buffer system wherein the reservoir buffer is ofa different pH and ionic strength from the buffer used to castthe gel. The SDS-polypeptide complexes in the sampleapplied to the gel are swept along by a moving boundarycreated when an electric current is passed between theelectrodes. After migrating through the stacking gel of highporosity, complexes get deposited in a very thin zone on thesurface of the resolving gel. On further electrophoresis,polypeptides get resolved based on their size in the resolvinggel.

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Western Blotting:

Blotting is transfer of resolved proteins from the gel ontoa surface of a suitable membrane, done commonly byelectrophoresis and referred to as electroblotting. The gel

is placed in contact with nitrocellulose membrane which isthen sandwiched between filter paper, two porous pads andtwo plastic supports. The entire set up is then placed in anelectrophoretic tank containing blotting buffer. The proteinsget transferred to the corresponding position on the membraneas resolved on the polyacrylamide gel, forming a mirror imageof the gel. Protein of interest on the membrane is furtherlocated by immunodetection.

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Immunodetection

The transferred proteins bound to the surface of nitrocellulosemembrane are detected using immunological reagents. Thisprocess is known as immunodetection . All the unoccupied

sites on the membrane are first blocked with either an inertprotein, a detergent or any other suitable blocking agent.The membrane is then probed with a primary antibody specificto the protein of interest. The Ag-Ab complex formed on themembrane is then identified using an enzyme-labeledsecondary antibody and a suitable substrate to the enzyme,which results in a coloured band on the nitrocellulosemembrane, referred to as blot development (Refer Fig 2).

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Gel

NC Membrane

Electroblotting

NC Membrane

Gel

(a) (b)

NCmembrane

Fig 1: Transfer of protein from gel to membrane

(a) Membrane is in close contact with polyacrylamide gelcontaining proteins for electroblotting.

(b) At the end of electrotransfer, all proteins migrate tonitrocellulose (NC) membrane.

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Kit Description:

In this kit, bacterial lysate havingGlutathione-S-Transferase (GST) fusion protein is provided,which will be electrophoresed in duplicates along with a

standard protein marker on a polyacrylamide gel. Followingelectrophoresis, the lysate and marker will be stained toknow the electrophoretic mobility of the GST fusion protein,while the other electrophoresed lysate sample will betransferred by electroblotting onto nitrocellulose membrane.The electroblotted sample will then be detected using anti-GST IgG as primary antibody and secondary antibody labeledwith Horse Radish Peroxidase (HRP). HRP is then detectedusing hydrogen peroxide as a substrate andTetramethylbenzidine (TMB) as a chromogen. HRP acts onhydrogen peroxide to release oxygen, which oxidizes theTMB to TMB oxide. The TMB oxide is deposited whereverenzyme is present and appears as a blue band on the NCmembrane.

KT21 : Kit is designed to carry out 5 Western Blottingexperiments. The kit also includeselectrophoresis equipment with accessories(ETS-3) required for electrophoresis.

KT21A : Kit is designed to carry out 5 Western Blottingexperiments.

KT21B : Kit is designed to carry out 20 WesternBlotting experiments.

Note : Electrophoresis equipment is required forKT21A and KT21B.

Duration of experiment:Experiment is carried out over aspan of two days, approximate time taken on each day is

indicated below.Day 1:6-8 hours (SDS-PAGE and Electroblotting)

Day 2:3 hours (Immunodetection, Observation and Results)

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Protein (Ag) on the membrane

Blocking the unoccupied sites onthe membrane with inert protein

Incubate with primary antibody[ ] and wash

Incubate with secondary antibody- enzyme conjugate [ ] andwash

Incubate with substrate[ ]and

wash

NC Membrane

Protein of interest seen as acoloured band.

Fig 2: Schematic Representation of Blot development.

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8 9

Materials Provided:

The list below provides information about the materialssupplied in the kit. The products should be stored assuggested. Use the kit within 6 months of arrival.

Quantity

Materials KT21/21A KT21B Store

(5 expts.) (20 expts.)

SDS Separating gel mix 40 ml 125 ml 4C

SDS Stacking gel mix 20 ml 50 ml 4C

Ammonium persulphate 2 x 100 mg 4 x 100 mg RT

(APS)

10X Reservoir buffer 125 ml 2 x 250 ml 4C

Sample loading buffer 0.5 ml 2.0 ml 4C

Protein marker 0.125 ml 0.5 ml 4C

Protein samples 5 Nos. 20 Nos. 4C

20X Blotting buffer 125 ml 2 x 250 ml RTcomponent A

20X Blotting buffer 125 ml 2 x 250 ml 4Ccomponent B

Blocking agent 2 g 8 g 4C

10X Diluent buffer 5 ml 25 ml 4C

10X Assay buffer 10 ml 50 ml 4C

25X Wash buffer 10 ml 50 ml 4C

Primary antibody 5 Nos. 20 Nos. 4C

1000X HRP conjugate 0.15 ml 0.25 ml 4C

10X TMB/H2O

25 ml 20 ml 4C

Nitrocellulose (NC)

membrane with 5 Nos. 20 Nos. RTFilter Paper

Ezee Blue 125 ml 2 x 250 ml RT

Materials Required:

Equipment : Gel Rocker (optional)Glassware : Conical flask, Measuring cylinder,

Petri dish, Staining tray.

Reagent : Distilled water.Other Requirements: Micropipettes, Tips, Water bath.

Note:

Read the entire procedure before starting theexperiment.

Wear gloves while handling the gel and membrane.

Prepare 1X TMB/H2O

2, 1X secondary antibody, blocking

bufferjust before use.

Resuspend an aliquot of APS in 1 ml of distilled water.Store at 4C. Use within 2 months.

Destaining step is not required on staining the gel withEzee blue stainer (Coommassie blue).

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Prepare the reagents as indicated below before startingeach experiment:

Preparation of 1X Assay Buffer: To 2 ml of 10X assaybuffer add 18 ml of distilled water to get 20 ml of 1X assay

buffer.Preparation of Protein Sample: Resuspend proteinsample in 25 l of distilled water.

Preparation of Primary Antibody: Resuspend an aliquotof primary antibody in 1 ml of 1X assay buffer. Transfer to atest tube and make up the volume to 10 ml with 1X assaybuffer.

Preparation of Blotting Buffer: Mix 25 ml each of blottingbuffer components A and B with 450 ml of distilled water.

Preparation of 1X Reservoir Buffer: To 25 ml of 10XReservoir Buffer add 225 ml of distilled water to get 250 ml of1X resevoir buffer.

Preparation of 1X Secondary Antibody: Pipette 10 l of1000X HRP conjugate and add 9.90 ml of 1X assay buffer.Mix thoroughly.

Preparation of Diluent Buffer: Dilute 1 ml of 10X diluentbuffer to 10 ml with distilled water just before use.

Preparation of Blocking Buffer: Weigh 300 mg of blockingagent, suspend in 10 ml of 1X diluent buffer.

Procedure:

Day 1:SDS-PAGE

1. Assemble the plates for casting gel as shown below.

2. Clamp the assembly of plates to fix it in a gel castingapparatus. Ensure the assembly is leak proof by fillingwater between the plates. Silicon grease can be appliedto spacer to make a water-tight seal.

3. Add 50 l of APS solution to 5 ml of SDS separatinggel mix and pour the gel solution between the plates tillthe level is 2 cm below the top edge of notched plate.

4. Add 200 to 250 l of water to make the surface even.

5. After the gel is set (approximately 20-30 min.), washthe top of the separating gel with distilled water anddrain off the water completely.

6. Add 20 l of APS solution to 2 ml of stacking gel mixand pour directly onto the polymerized separating gel.

7. Insert the comb into the gel solution carefully withouttrapping any bubbles, about 1cm above the separatinggel. The stacking gel will set in approximately 10 min.

8. Add 25 l of sample loading buffer to protein sample.

9. Add 25 l of sample loading buffer to 25 l of protein

marker.10. Place it in a boiling water bath for 5 minutes.

Base plate

Notched

Spacers

Assembled for casting GelGlass Plates & Spacers

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21. Decant the staining solution add minimum quantity ofwater to cover the gel.

Note:Cover the tray and leave it overnight at roomtemperature.

Electroblotting:22. Assemble the blotting sandwich within the blotting

cassette as shown in the figure. Take care to avoid airbubbles between the gel and NC membrane.

23. Insert the cassette into the apparatus filled with blottingbuffer and connect blotting unit to power supply as perthe convention,

red: anode, black: cathode

24. Electrophorese the sample at 50 V for 2 hours for blottingto occur.

25. Remove the NC membrane gently from the cassetteand place the membrane in 10 ml of freshly preparedblocking buffer taken in a petri dish. Leave it overnightat 4C.

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Anode cassette cover

Cathode cassette cover

Sponge

Sponge

Filter Paper

Filter Paper

Polyacrylamide GelNC Membrane

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11. After the stacking gel has set, carefully remove thecomb and the bottom spacer. Wash the wellsimmediately with distilled water to removenon-polymerized acrylamide. Fill the bottom reservoirwith 1X reservoir buffer and carefully fix the plate to the

apparatus without trapping any air bubbles between thebuffer and the bottom of the gel. Fix the plates to PAGEapparatus. Fill the top reservoir with 1X reservoir buffer.

12. Load 30 l protein marker in well 1, 40 l of proteinsample in well 2 and 5 l of protein sample in well 4.Note down the order of loading. Connect the cords tothe power supply accroding to the conventionred: anode, black: cathode.

13. Set voltage at 100 V and switch on the power supply.

14. When the dye front comes to 0.5 cm above the bottomof the gel, turn off the power. This will take approximately1 to 11/

2hours.

15. Remove the gel plates and gently pry the plates apart

using a spatula or similar tool, not at the notch.16. Transfer the gel to a tray containing water, wash thegel for 1-2 minutes at room temperature.

17. Decant water, cut the gel along lane 3.

18. Transfer lane 4 i.e., protein sample in 10 ml of blottingbuffer taken in a petri dish. Keep at room temperaturefor 10 minutes. Following incubation, proceed forelectroblotting as described in step 22.

19. To the gel piece (lanes 1 & 2) add minimum of20 ml water.

20. Decant the water, add minimum 20 ml of Ezee BlueStain. Stain at room temperature for 1-2 hours.

Note: For uniform staining and washing, place thetray on a rocker or shake intermittently every 10to 15 minutes.

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Day 2: Immunodetection

26. Discard blocking buffer.

27. Immerse blot in 10 ml of primary antibody solution andmix gently for 30 minutes.Discard the primary antibody

solution.28. Wash the blot by immersing in 10 ml wash buffer for3-5 minutes. Repeat the wash two times. Discard thebuffer each time.

29. Immerse the blot in 10 ml of 1X HRP labeled antibody.Mix gently for 30 minutes. Discard the HRP labeledantibody.

30. Wash the blot by immersing in 10 ml wash buffer for3-5 minutes. Repeat the wash process four to five times.discarding the buffer each time.

31. Immerse the washed blot in 10 ml of substrate solution,mix gently for 5-10 minutes, within this time colouredband will appear.

32. Remove the blot, wash with distilled water, discard and

dry.Note: Although the colored band fades with time,the rate of color loss can be retarded if the blotsare kept in dark.

33. Compare the SDS-Polyacrylamide gel with thedeveloped NC membrane.

Observation: On staining SDS-Polyacrylamide gel, different proteins

will appear as dark blue bands against a light bluebackgroud.

On immunodetection, a single blue band will be observedon NC membrane.

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Interpretation:

On electrophoresis of bacterial lysate on SDS-PAGE, manybands indicating the different proteins present in the crudesample are seen. A predominant band among these is that

of GST fusion protein corresponding to 26 kD protein of themarker. Following transfer and immunodetection, oneobserves a predominant band corresponding to GST proteinbound to anti GST antibody. However, few light bands maybe seen indicating the proteins to which anti-GST antibodycross reacts.

Fig: 3a Fig: 3b

Fig 3a: Protein bands as detected by Coommassie bluestaining of SDS-Polyacrylamide gel

Lane 1. Bacterial Lysate

Lane 2. Protein Marker - Standard proteins ranging inmolecular weight from 66 kD - 14.3 kD

Fig 3b: Immunodetection on blotted NC membrane.

Molecular Weight

66,000 Da

43,000 Da

29,000 Da

14,300 Da

The band on the nitrocellulose membrane indicates the

GST protein detected by the antibody (anti-GST) The position of the band on the membrane indicates its

electrophoretic mobility during electrophoresis.

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Ordering Information

Product Size Cat #

GeNeiTM Western Blotting 1 Pack KT21Teaching Kit(Consumables for 5 Experiments& Elpho Kit (ETS-3))

GeNeiTM Western Blotting 1 Pack KT21ATeaching Kit(Consumables for 5 Experiments)

GeNeiTM Western Blotting 1 Pack KT21BTeaching Kit(Consumables for 20 Experiments)

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Customer Support:[email protected]

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