Western Blotting
Content
Introduction .............................................................................................................................................2
Western Blotting Procedure .....................................................................................................................2
Preparation of Protein Samples ................................................................................................................2
Using the Vertical Gel System .................................................................................................................3
Using the Dry System Transferring ..........................................................................................................4
Blocking ...................................................................................................................................................6
Protein Detection ......................................................................................................................................6
Visualization of Chemiluminescent Detection .........................................................................................7
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Western Blotting
Introduction
Western Blotting (or immunoblotting) is a standard laboratory procedure allowing investigators
to verify the expression of a protein, determine the relative amount of the protein present in different
samples, and analyze the results of co-immunoprecipitation experiments. In this method, a target
protein is detected with gel system and a specific primary antibody in a given sample of tissue
homogenate or extract. Protein separation according to molecular weight is achieved using
denaturing SDS-PAGE. After transfer to a membrane, the target protein is probed with a specific
primary antibody and detected by chemiluminescence.
Western Blotting Procedure
Preparation of Protein Samples
Fig.1: Preparation of Protein Samples
- Cell culture is washed several times with PBS (Phosphate buffered saline).
* For help to maintain a constant pH.
- Culture is centrifuged at 2500 rpm for 5 minutes.
*For the cell precipitate.
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Western Blotting
-150 uL Protein Extraction Reagent is added.
* The purification of extracted proteins from a starting sample of cells or tissue.
-Waiting 5 minutes at room temperature.
-Centrifuged at 15000rpm for 5 minutes.
*Collecting protein from supernatant.
-Mixing protein with LDS (lithium dodecyl Sulfate) sample buffer.
* Samples at least 1:4 with sample buffer
* LDS Sample Buffer (4X) is used to prepare protein samples for denaturing gel
electrophoresis with the gels.
- Heat on heating block at 95-1000C for 5 minutes.
* To break the 3-dimensional structure.
Fig.2:Breaking 3-dimensional structure of Protein
Using The Vertical Gel System
- Measure protein concentration for loading equal amount of protein.
-Loading 1X MES or 1XMOPS buffer loading to gel tank.
- Samples are loaded into wells in the gel. One lane is usually reserved for a marker or ladder, a
commercially available mixture of proteins having defined molecular weights, typically stained so as
to form visible, coloured bands.
*Samples containing multiple proteins require 10-15 ug of protein per well.
Purified
samples containing single or very few proteins require less (2-5 ug). Less is required for the
more sensitive process of silver stain (1 ug).
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Western Blotting
Fig.3: Vertical Gel System
- Run the gel at 200 V for 45-50 minutes.
* When voltage is applied along the gel, proteins migrate into it at different speeds
* Can be changed voltage according to the size of the gel.
*Allow migration to continue until the blue dye front is at the end of the glass plates,
but does not migrate off the gel.
Using the Dry System Transferring
-Remove the gel from the tank for transfer after completion of electrophoresis.
*There is no need for any pretreatment of the gel after electrophoresis. Wash the gel briefly
in deionized water to remove any small gel pieces attached to the gel.
- Place the anode stack and wet the desired blotting membrane with deionized water. Remove any air
bubbles using the blotting roller.
-Place the gel on the membrane and place the fitler paper on the gel. Remove any air bubbles using the
blotting roller.
- Place the Cathode Stack, Top without the tray on top of Metal Spacer 2 with the copper electrode
side facing up.
*Ensure that all layers are aligned to the right to perform efficient de-bubbling.
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Western Blotting
Fig.4: Dry System Transferring
- Place the disposable sponge on the inner side of the lid.
* The sponge absorbs any excess liquid generated during blotting and exerts an even
pressure on the stack surface.
- Close the iBlot Lid and secure the latch. The red light is on indicating a closed circuit. Ensure the
correct program is selected. Select the appropriate program.
- Press the Start/Stop button to start the transfer. The red status light changes to green. The transfer
continues using the parameters that you programmed as described.
- The iBlot Gel Transfer Device signals the end of transfer with repeated beeping sounds, and flashing
green light and digital display. Press and release the Start/Stop button to stop the beeping. The light
turns to a steady red light.
* System was completed in 7 minutes.
- Transfer the proteins to a nitrocellulose membrane .Open the lid of the iBlot device. Collect to all
meterial from the device.
* Discard the iBlot disposable sponge and iBlot cathode stack, Top.
* Carefully remove and discard the gel and filter paper. Remove the nitrocellulose
membrane from the stack and proceed with the blocking procedure or stain the
membrane.
* Discard the iBlot anode stack, bottom.
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Western Blotting
Blocking
-Since the membrane has been chosen for its ability to bind protein and as both antibodies and the
target are proteins, steps must be taken to prevent interactions between the membrane and the
antibody used for detection of the target protein.
- Transferring the membrane from deionized water to PBS-T with non-fat dry milk(5% non-fat dry
milk in TBS). Waiting 30 minutes on the shaker.
* Blocking of non-specific binding is achieved by placing the membrane in a dilute solution
of protein - typically non-fat dry milk in Tris-Buffered Saline (TBS), with a minute
percentage of detergent such as Tween 20 or Triton X-100.
Fig.5: Blocking the membrane
Protein Detection
- Incubate the diluted primary antibody (1:250 to 1:1000 in blocking buffer) with membrane for 1 h in
blocking buffer on the shaker at room temperature.
- Wash the blot three times, 10 min in washing buffer (TBS containing 0.1% Tween 20) with shaking.
*After rinsing the membrane to remove unbound primary antibody, the membrane is
exposed to another antibody, directed at a species-specific portion of the primary
antibody.
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Western Blotting
- Incubate blot with secondary antibody(anti-rabbit IgG-HRP conjugate) (diluted 1:10,000 -1:2,000 in
blocking buffer) for 1 h in blocking buffer at room temperature.
- Wash three times, 10 min in washing buffer with shaking.
*After the unbound probes are washed away, the western blot is ready for detection of the
probes that are labeled and bound to the protein of interest.
Fig.6: Washing the membrane
Visualization of Chemiluminescent Detection
- Chemiluminescent detection methods depend on incubation of the western blot with a substrate that
will luminesce when exposed to the reporter on the secondary antibody.
*The last step works at darkroom.
-10 ml Detection Solution according to the package insert provided with the Chemiluminescence
Blotting Substrate reagent set. Cover the blot membrane with Detection Solution and incubate the
membrane for 20 seconds.
-Drain excess Detection Solution from the membrane, and wrap the blot in plastic. Place a sheet of Xray film over the plastic wrapped membrane in a film cassette.
* Make sure the protein side of the membrane faces the X-ray film. This ensures maximum
sensitivity.
-Close the cassette and expose the membrane to the X-ray film for 20 s.
* Incubation time will vary with the intensity of the protein bands and the type of
film used. Use 20 seconds as a starting point for a typical experiment.
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Western Blotting
-Remove the X-ray film from the cassette and develop the film according to the manufacturer’s
instructions.
*If the first film is too dark (overexposed),decrease the exposure time to 10–20 s.
*If the first film is too light (underexposed),increase the exposure time to 20–60 s.
Fig.7: Western Blot X-ray film
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