Western Blotting

GeNeiTM

Western Blotting

GeNeiTM

GeNeiTM Western Blotting Teaching Kit Manual

Cat No. KT21 KT21A KT21B

Bangalore Genei, 2007 Bangalore Genei, 2007

New Cat No. 106129 106160 106161

Revision No.: 00260705

Western Blotting

GeNeiTM

Western Blotting

GeNeiTM
CONTENTS
Page No.

Objective Principle Kit Description Materials Provided Procedure Observation & Interpretation
ORDERING INFORMATION

3 3 5 7 11 14
16

Bangalore Genei, 2007

Bangalore Genei, 2007

Western Blotting

GeNeiTM

Western Blotting

GeNeiTM

Objectives:
To learn the technique of Western Blotting which involves the 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 or immunoblotting) is a rapid and sensitive assay for detection and characterization of proteins. Western Blotting technique exploits the inherent specificity of antigen-antibody interaction to identify specific antigens by polyclonal or monoclonal antibodies. SDS-PAGE: Sodium Dodecyl Sulphate - Polyacrylamide Gel Electrophoresis (SDS-PAGE ) is carried out in a discontinuous buffer system wherein the reservoir buffer is of a different pH and ionic strength from the buffer used to cast the gel. The SDS-polypeptide complexes in the sample applied to the gel are swept along by a moving boundary created when an electric current is passed between the electrodes. After migrating through the stacking gel of high porosity, complexes get deposited in a very thin zone on the surface of the resolving gel. On further electrophoresis, polypeptides get resolved based on their size in the resolving gel.

Bangalore Genei, 2007

Bangalore Genei, 2007

Western Blotting

GeNeiTM

Western Blotting

GeNeiTM

Western Blotting: Blotting is transfer of resolved proteins from the gel onto a surface of a suitable membrane, done commonly by electrophoresis and referred to as electroblotting. The gel is placed in contact with nitrocellulose membrane which is then sandwiched between filter paper, two porous pads and two plastic supports. The entire set up is then placed in an electrophoretic tank containing blotting buffer. The proteins get transferred to the corresponding position on the membrane as resolved on the polyacrylamide gel, forming a mirror image

of the gel. Protein of interest on the membrane is further located by immunodetection.

Gel Gel Electroblotting
NC membrane

Immunodetection The transferred proteins bound to the surface of nitrocellulose membrane are detected using immunological reagents. This process is known as immunodetection. All the unoccupied sites on the membrane are first blocked with either an inert protein, a detergent or any other suitable blocking agent. The membrane is then probed with a primary antibody specific to the protein of interest. The Ag-Ab complex formed on the membrane is then identified using an enzyme-labeled secondary antibody and a suitable substrate to the enzyme, which results in a coloured band on the nitrocellulose membrane, referred to as blot development (Refer Fig 2).

NC Membrane NC Membrane

(a)

(b)

Fig 1: Transfer of protein from gel to membrane (a) Membrane is in close contact with polyacrylamide gel containing proteins for electroblotting. (b) At the end of electrotransfer, all proteins migrate to nitrocellulose (NC) membrane.

Bangalore Genei, 2007

Bangalore Genei, 2007

Western Blotting
Protein (Ag) on the membrane

GeNeiTM

Western Blotting

GeNeiTM

Kit Description:
In this kit, bacterial lysate having Glutathione-S-Transferase (GST) fusion protein is provided, which will be electrophoresed in duplicates along with a standard protein marker on a polyacrylamide gel. Following electrophoresis, the lysate and marker will be stained to know the electrophoretic mobility of the GST fusion protein, while the other electrophoresed lysate sample will be transferred by electroblotting onto nitrocellulose membrane. The electroblotted sample will then be detected using antiGST IgG as primary antibody and secondary antibody labeled with Horse Radish Peroxidase (HRP). HRP is then detected using hydrogen peroxide as a substrate and Tetramethylbenzidine (TMB) as a chromogen. HRP acts on hydrogen peroxide to release oxygen, which oxidizes the TMB to TMB oxide. The TMB oxide is deposited wherever enzyme is present and appears as a blue band on the NC membrane. KT21 : Kit is designed to carry out 5 Western Blotting experiments. The kit also includes electrophoresis equipment with accessories (ETS-3) required for electrophoresis. KT21A : KT21B : Note : Kit is designed to carry out 5 Western Blotting experiments. Kit is designed to carry out 20 Western Blotting experiments.

Blocking the unoccupied sites on the membrane with inert protein Incubate with primary antibody [ ] and wash Incubate with secondary antibody - enzyme conjugate [ ] and wash Incubate with substrate[ ]and wash

NC Membrane

Protein of interest seen as a coloured band. Fig 2: Schematic Representation of Blot development.

Electrophoresis equipment is required for KT21A and KT21B. Duration of experiment: Experiment is carried out over a span 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)
Bangalore Genei, 2007

Bangalore Genei, 2007

Western Blotting

GeNeiTM

Western Blotting

GeNeiTM

Materials Provided:
The list below provides information about the materials supplied in the kit. The products should be stored as suggested. Use the kit within 6 months of arrival.
Quantity Materials SDS Separating gel mix KT21/21A
(5 expts.)

Materials Required:
Equipment : Gel Rocker (optional) Glassware : Conical flask, Measuring cylinder, Petri dish, Staining tray. Reagent : Distilled water. Other Requirements: Micropipettes, Tips, Water bath.

KT21B
(20 expts.)

Store

Note:
4C 4C RT 4C 4C 4C 4C RT 4C 4C 4C 4C 4C 4C 4C 4C RT RT
Bangalore Genei, 2007

40 ml

125 ml

SDS Stacking gel mix 20 ml 50 ml Ammonium persulphate 2 x 100 mg 4 x 100 mg (APS) 10X Reservoir buffer 125 ml 2 x 250 ml Sample loading buffer 0.5 ml 2.0 ml Protein marker 0.125 ml 0.5 ml Protein samples 5 Nos. 20 Nos. 20X Blotting buffer 125 ml 2 x 250 ml component A 20X Blotting buffer 125 ml 2 x 250 ml component B Blocking agent 2g 8g 10X Diluent buffer 5 ml 25 ml 10X Assay buffer 10 ml 50 ml 25X Wash buffer 10 ml 50 ml Primary antibody 5 Nos. 20 Nos. 1000X HRP conjugate 0.15 ml 0.25 ml 5 ml 20 ml 10X TMB/H2O2 Nitrocellulose (NC) membrane with 5 Nos. 20 Nos. Filter Paper Ezee Blue 125 ml 2 x 250 ml
Bangalore Genei, 2007

Read the entire procedure before starting the experiment. Wear gloves while handling the gel and membrane. Prepare 1X TMB/H2O2, 1X secondary antibody, blocking buffer just 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 with Ezee blue stainer (Coommassie blue).

Western Blotting

GeNeiTM

Western Blotting

GeNeiTM

Prepare the reagents as indicated below before starting each experiment: Preparation of 1X Assay Buffer: To 2 ml of 10X assay buffer add 18 ml of distilled water to get 20 ml of 1X assay buffer. Preparation of Protein Sample: Resuspend protein sample in 25 l of distilled water. Preparation of Primary Antibody: Resuspend an aliquot of primary antibody in 1 ml of 1X assay buffer. Transfer to a test tube and make up the volume to 10 ml with 1X assay buffer. Preparation of Blotting Buffer: Mix 25 ml each of blotting buffer components A and B with 450 ml of distilled water. Preparation of 1X Reservoir Buffer: To 25 ml of 10X Reservoir Buffer add 225 ml of distilled water to get 250 ml of 1X resevoir buffer. Preparation of 1X Secondary Antibody: Pipette 10 l of 1000X HRP conjugate and add 9.90 ml of 1X assay buffer. Mix thoroughly. Preparation of Diluent Buffer: Dilute 1 ml of 10X diluent buffer to 10 ml with distilled water just before use. Preparation of Blocking Buffer: Weigh 300 mg of blocking agent, suspend in 10 ml of 1X diluent buffer.

Procedure:
Day 1: SDS-PAGE 1. Assemble the plates for casting gel as shown below.
Glass Plates & Spacers Base plate Notched Assembled for casting Gel

Spacers

2. Clamp the assembly of plates to fix it in a gel casting apparatus. Ensure the assembly is leak proof by filling water between the plates. Silicon grease can be applied to spacer to make a water-tight seal. 3. Add 50 l of APS solution to 5 ml of SDS separating gel mix and pour the gel solution between the plates till the 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.), wash the top of the separating gel with distilled water and drain off the water completely. 6. Add 20 l of APS solution to 2 ml of stacking gel mix and pour directly onto the polymerized separating gel. 7. Insert the comb into the gel solution carefully without trapping any bubbles, about 1cm above the separating gel. 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.
Bangalore Genei, 2007

Bangalore Genei, 2007

10

11

Western Blotting

GeNeiTM

Western Blotting

GeNeiTM

11. After the stacking gel has set, carefully remove the comb and the bottom spacer. Wash the wells immediately with distilled water to remove non-polymerized acrylamide. Fill the bottom reservoir with 1X reservoir buffer and carefully fix the plate to the apparatus without trapping any air bubbles between the buffer and the bottom of the gel. Fix the plates to PAGE apparatus. Fill the top reservoir with 1X reservoir buffer. 12. Load 30 l protein marker in well 1, 40 l of protein sample in well 2 and 5 l of protein sample in well 4. Note down the order of loading. Connect the cords to the power supply accroding to the convention red: 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 bottom of the gel, turn off the power. This will take approximately 1 to 11/2 hours. 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 the gel 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 blotting buffer taken in a petri dish. Keep at room temperature for 10 minutes. Following incubation, proceed for electroblotting as described in step 22. 19. To the gel piece (lanes 1 & 2) add minimum of 20 ml water. 20. Decant the water, add minimum 20 ml of Ezee Blue Stain. Stain at room temperature for 1-2 hours. Note: For uniform staining and washing, place the tray on a rocker or shake intermittently every 10 to 15 minutes.
Bangalore Genei, 2007

21. Decant the staining solution add minimum quantity of water to cover the gel. Note:Cover the tray and leave it overnight at room temperature. Electroblotting: 22. Assemble the blotting sandwich within the blotting cassette as shown in the figure. Take care to avoid air bubbles between the gel and NC membrane.
Anode cassette cover Sponge Filter Paper Polyacrylamide Gel Filter Paper Sponge Cathode cassette cover

NC Membrane

23. Insert the cassette into the apparatus filled with blotting buffer and connect blotting unit to power supply as per the convention, red: anode, black: cathode 24. Electrophorese the sample at 50 V for 2 hours for blotting to occur. 25. Remove the NC membrane gently from the cassette and place the membrane in 10 ml of freshly prepared blocking buffer taken in a petri dish. Leave it overnight at 4C.

12

Bangalore Genei, 2007

13

Western Blotting

GeNeiTM

Western Blotting

GeNeiTM

Day 2: Immunodetection 26. Discard blocking buffer. 27. Immerse blot in 10 ml of primary antibody solution and mix gently for 30 minutes.Discard the primary antibody solution. 28. Wash the blot by immersing in 10 ml wash buffer for 3-5 minutes. Repeat the wash two times. Discard the buffer each time. 29. Immerse the blot in 10 ml of 1X HRP labeled antibody. Mix gently for 30 minutes. Discard the HRP labeled antibody. 30. Wash the blot by immersing in 10 ml wash buffer for 3-5 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 coloured band 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 blots are kept in dark. 33. Compare the SDS-Polyacrylamide gel with the developed NC membrane.

Interpretation:
On electrophoresis of bacterial lysate on SDS-PAGE, many bands indicating the different proteins present in the crude sample are seen. A predominant band among these is that of GST fusion protein corresponding to 26 kD protein of the marker. Following transfer and immunodetection, one observes a predominant band corresponding to GST protein bound to anti GST antibody. However, few light bands may be seen indicating the proteins to which anti-GST antibody cross reacts. 1 2
Molecular Weight 66,000 Da 43,000 Da 29,000 Da 14,300 Da

Fig: 3a Fig: 3b Fig 3a: Protein bands as detected by Coommassie blue staining of SDS-Polyacrylamide gel Lane 1. Bacterial Lysate Lane 2. Protein Marker - Standard proteins ranging in molecular weight from 66 kD - 14.3 kD Fig 3b: Immunodetection on blotted NC membrane. 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. Bangalore Genei, 2007 15

Observation:
On staining SDS-Polyacrylamide gel, different proteins will appear as dark blue bands against a light blue backgroud. On immunodetection, a single blue band will be observed on NC membrane.

Bangalore Genei, 2007

14

Western Blotting

GeNeiTM

Western Blotting

GeNeiTM

Ordering Information
Product GeNeiTM Western Blotting Teaching Kit (Consumables for 5 Experiments & Elpho Kit (ETS-3)) GeNeiTM Western Blotting Teaching Kit (Consumables for 5 Experiments) GeNeiTM Western Blotting Teaching Kit (Consumables for 20 Experiments) Email: Sales: geneisales@sanmargroup.com Customer Support: geneitechsupport@sanmargroup.com Size 1 Pack Cat # KT21

Note:

1 Pack

KT21A

1 Pack

KT21B

Bangalore Genei, 2007

16

Bangalore Genei, 2007