Western Blot- Electrophoresis & Transfer Protocol

Learn more about western blot, please see Previous Section.

Sample Loading of Western Blot

Loading buffer:

Laemmli (Tris-glycine) buffering systems are the most commonly used and are comprised of a stacking gel of pH 6.8 and a resolving gel of between pH 8 and 9. One potential drawback of this popular system is that disulfide bonds tend to form between cysteine residues at this relatively high pH, although this problem can be alleviated by the addition of a reducing agent to the sample. In addition, we usually add 0.1mg bromophenol blue as indicator into the sample loading buffer. Use special gel loading tips or a micro-syringe to load the complete sample in a narrow well. Take care not to pierce the base of the well with the tip as this will create a distorted band. Never overfill wells, this could lead to poor data if samples spill into adjacent wells and poorly resolved bands. Load 20-40 μg total protein per mini-gel well. The gels will be submerged in migration buffer which normally contains SDS, except in native gel electrophoresis. A standard migration buffer (also called running buffer) for PAGE is 1 X Tris-glycine.

Molecular weight markers:

Molecular weight markers are used to define the size of proteins run in a gel. Markers are composed of different proteins of known size and the distances migrated over the time course of the run provide a logarithmic scale by which to estimate the size of unknown proteins. For most runs, it is convenient to reserve at least one separate lane on the gel to run the molecular weight markers.

Loading controls:

Loading controls are required to check that the lanes in your gel have been evenly loaded with sample. This is important especially when a comparison must be made between the expression levels of a protein in different samples (Table 1). They are also useful to check for even transfer from the gel to the membrane across the whole gel. Where even loading or transfer have not occurred, the loading control bands can be used to quantify the protein amounts in each lane.

Table 1. Common used loading controls

Protein Transfer of Western Blot

Visualization of proteins in gels (Optional):
Before protein transfer, we could validating the electrophoresis result by visualization of proteins in gel. The visualization of protein at this stage is useful to determine if proteins have migrated uniformly and evenly. Use the copper stain if you plan to transfer the separated proteins to a membrane, as the Coomassie stain is not reversible.

After completion of the separation of proteins by polyacrylamide gel electrophoresis (PAGE), the next step is to transfer the proteins from the gel to a solid support membrane for further analysis. This membrane is usually made of nitrocellulose (NC) or polyvinylidene fluoride (PVDF). PVDF membranes require careful pre-treatment: Soak it in methanol for 1-2 minutes. Incubate in ice cold transfer buffer for 5 minutes. The gel needs to equilibrate for 3-5 minutes in ice cold transfer buffer. Failure to do so will cause shrinking while transferring, and a distorted pattern of transfer (Table 2).

Table 2. The comparison of NC and PVDF membrane.

Electro-transfer is almost exclusively the contemporary transfer method of choice due to its speed, uniformity of transfer, and transfer efficiency. Electro-transfer relies on the same electro-mobility principles that drive the migration of proteins during separation in PAGE. The gel, membrane, and electrodes are assembled in a sandwich so that proteins move from gel to membrane, where they are captured, in a pattern that perfectly mirrors their migration positions in the gel. The two commonly used electro-transfer techniques are wet transfer and semi-dry transfer (Figure 1). Both are based on the same principles and differ only in the mechanical devices used to hold the gel/membrane stack and applications of the electrical field.

Figure 1. Two electro-transfer techniques: wet transfer and simi-dry transfer.

Wet transfer:

In this choice of transfer, the gel and membrane are both fully immersed in transfer buffer and a current is applied in the direction of the gel to the membrane. Generally, wet transfer requires cooling of the unit and internal recirculation of the transfer buffer by the presence of a stirring magnet. Wet transfer is recommended for large proteins, but it is a relatively slow technique, requiring large volumes of buffer.

Reference protocol of wet transfer:

1. Cut away the stacking gel and cut one corner from the resolving gel so that enable you to correctly orientate the gel if it “flips over” during equilibration.
2. Pre-wet and equilibrate the membrane in transfer buffer.
   • PVDF membranes need to be pre-wetted in methanol and water before equilibration in transfer buffer.
   • Always wear gloves when handling membranes to avoid fingerprints which will negatively affect the results.
3. Pre-wet a sponge and place it on the submerged part of the cassette. Press gently to expel all air bubbles. Place two pre-wetted blotting papers on to the sponge. Place the membrane on top of the blotting papers. Place the gel on top of the membrane. Place two additional pre-wetted blotting papers on the gel. Gently pressing to remove air bubbles. Finally place a pre-wetted sponge on top of the stack and close the cassette.
4. Place the cassette in the transfer tank. Watch out the orientation. The membrane should be closest to the anode (+) as the protein with negative charge would move towards the anode.
5. Connect the transfer tank to the power supply and transfer according to the recommendations of the manufacturer. (Transfer condition: 30V overnight transfer or 100V for 1h transfer)

Semidry transfer:

Semidry transfer is faster than wet transfer and consumes less buffer. The membrane is placed in direct contact with the gel and several layers of filter paper soaked in transfer buffer are placed above and below the gel and membrane. The filter paper->gel->membrane->filter paper layers are then sandwiched between two plates that form an anode (+) and a cathode (-) when an electric field is applied. Semidry transfer is usually less efficient than wet transfer, especially for large proteins. Heating is less of a problem with semidry transfer but semidry systems should be avoided for extended transfer times as this may lead to overheating and gel drying due to buffer depletion.

Reference protocol of wet transfer:

1. Cut away the stacking gel and cut one corner from the resolving gel so that enable you to correctly orientate the gel if it “flips over” during equilibration.
2. Cut blotting paper to the same size as the gel or slightly smaller. Saturate blotting paper with transfer buffer one by one and remove all trapped air gently.
3. Pre-wet and equilibrate the membrane in transfer buffer. Place the pre-wetted membrane onto the stack of wetted blotting paper. Place the gel on the membrane. Cover the gel with layers of saturated blotting paper. Remove any trapped air gently.
4. Connect to the power supply and transfer according to the recommendations of the manufacturer.

Notes on transfer of large and small proteins:

For large proteins (Mr>100 000). Be sure to run your samples in a low-concentration gel, 8% or less. These will be very fragile, so handle carefully. Large proteins will tend to precipitate in the gel, hindering transfer. Adding SDS to a final concentration of 0.1% in the transfer buffer will discourage this. Methanol tends to remove SDS from proteins, so reducing the methanol percentage to 10% or less will also guard against precipitation. Lowering methanol in the transfer buffer also promotes swelling of the gel, allowing large proteins to transfer more easily. Methanol is only necessary if using nitrocellulose. If using PVDF, methanol can be removed from the transfer buffer altogether, and is only needed to activate the PVDF before assembling the gel/membrane sandwich. Choose wet transfer overnight at 4°C instead of semi-dry transfer.

For small proteins (Mr<100 000):

Remove SDS from transfer buffer. All proteins are hindered from binding to membranes by SDS, but this is especially true for small proteins. Keep the methanol content at 20%. This will help remove as much SDS as possible and improve transfer efficiency.

Learn more about western blot, please see Next Section.

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