Western Blot: Overview, Technique, Theory, and Trouble Shooting
Objective of Western Blot
Transfer of proteins from SDS-PAGE to solid supports and detection of immobilized proteins by antigen-antibody reaction by Western blotting.
Principle of Western Blot
Western blot process is to proteins what southern blotting is to DNA. In both techniques, electrophoretically separated components are transferred from Polyacrylamide gel electrophoresis (PAGE) to a solid support (nitrocellulose membrane) and probed with reagents that are specific for particular sequences of amino acids (western blot) or nucleotides (southern hybridization).
In the case of proteins probes usually are antibodies that react specifically with the antigenic epitopes displayed by the target protein attached to the solid support (nitrocellulose membrane).
Therefore, Western blot technique is extremely useful for the identification and quantitation of specific proteins in the complex mixtures of various proteins that are not radiolabeled.
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A variety of different solid supports have been used for western blotting such nitrocellulose membrane and Polyvinylidene fluoride or polyvinylidene difluoride (PVDF) membrane.
These include cyanogen-bromide-activated paper, PVDF (polyvinyledene difluoride) membrane, nitrocellulose filter etc.
Nowadays, most western blotting is carried out by direct electrophoretic transfer of proteins from the gel to a nitrocellulose filter.
Two types of electrophoretic apparatuses are available for electroblotting.
In the first one, one side of the gel is placed in contact with a piece of nitrocellulose filter and the polyacrylamide gel and its attached filter are then sandwiched between whatmann 3 mm paper, two porous pads and two plastic supports.
The entire set-up is then immersed in an electrophoresis tank containing Tris-glycine electrophoresis buffer at pH 8.3 and equipped with standard platinum electrodes.
The nitrocellulose paper is placed towards the anode.
An electric current is then passed for around 12 h during which the proteins get transferred from the gel to nitrocellulose filter.
In order to prevent over-heating and consequent formation of air-bubbles in the sandwich the transfer is carried out in cold conditions.
In a newer type of apparatus, the gel and its attached nitrocellulose filter are sandwiched between pieces of whatmann 3 mm paper that have been soaked in the transfer buffer containing Tris, glycine, SDS and methanol.
In this case since there is very little requirement of the electrophoresis buffer, the system is termed as semi-dry system resulting in semi-dry blotting.)
The sandwich is then placed between plate electrodes, with nitrocellulose filter on the anodic side.
Transfer of proteins can be carried out at room temperature and the time required is minimized to 1.5-2 h for complete transfer.
Western Blot Requirements
Transfer buffer: 39 mM glycine, 48 mM Tris base, 0.037% SDS and 20% methanol.
- Make the volume with distilled water.
- pH should be 8.3.
Tris Buffered saline (TBS) with containing 0.1% (v/v) Tween-20 (TBST)
Western Blot Procedure
1. When the SDS-PAGE is approaching the end of its run, rinse the plates with laboratory grade distilled water followed by wipe off any beads of liquid that adhere to them with nonabsorbent tissues using methanol.
2. Wearing gloves cut around 4 pieces of whatmann 1 mm paper and one piece of PVDF membrane to the exact size of the SDS-PAGE gel (if the whatmann paper or filter is of size larger than the polyacrylamide gel there is a good chance that the overhanging edges of the paper and the filter will touch causing a short circuit that will prevent the transfer of protein from the polyacrylamide gel to the filter). Mark one end of the filter with a soft lead pencil or cut the filter slightly on the lower left side.
3. To activate the PVDF membrane, soak it in absolute methanol for 1 minute. Thereafter, soak the PVDF membrane in transfer buffer.
4. Soak the pieces of whatmann paper in a shallow tray containing a small amount of transfer buffer.
5. Wearing gloves, set up the transfer apparatus as follows:
I) Place around 2 pieces of soaked whatmann paper on the bottom electrode.
II) Now very carefully place the soaked PVDF membrane on top of the whatmann paper carefully.
III) On top of the PVDF memberane carefully place the gel preventing entrapment of any air bubble.
IV) Place 2 pieces of soaked whatmann papers on top of the gel (At each step, stack the sheets on top of the other so that they are exactly aligned.) Using a spacer or a glass pipette as a roller, squeeze out air bubbles if any.
V) Place the upper electrode on top of the stack. Connect the electrical leads. Apply a constant current of 2.5 A for 7 min.
VI) After 7 min turn off the power supply and disconnect the leads. Disassemble the transfer apparatus from the top to downward, peeling off each layer in turn. Place the memberane in a tray containing Ponceau S for staining.
VII) Wash the membrane with distilled water. Block the non-specific binding by incubating the membrane with 1xTBS containing 2% BSA w/v for 1 h at room temperature.
VIII) After blocking, wash once with 1xTBS containing Tween 20 and incubate with primary antibody overnight at 4ᵒC. The antibody should be diluted with 1xTBS with 0.5% BSA (w/v) according to the supplier’s instructions. Once the incubation time is over, remove the primary antibody and wash the membrane three times with 1 x TBST
IX) After washing, incubate the membrane with secondary antibody which is conjugated with HRP (Horse Radish Peroxidase) and diluted in 01xTBS with 0.5% BSA for 1 hr at room temperature. Following the incubation, remove the antibody solution and wash three more times with 1 x TBST
X) Visualize protein expressions using the chemiluminescence detection substrate (Millipore) using chemidocumentation system.
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