Western blotting. Part III

Features

Patricia Leoni
Imperial College London

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https://doi.org/10.36866/pn.83.31

In the last of this series, Patricia Leoni discusses the process of transferring electropheresed proteins to a membrane (the ‘blot’) and the use of antibodies to identify proteins of interest from the many that present on the membrane.

The separation of proteins by electrophoresis on polyacrylamide gels does not provide any information on their identity. The proteins can be recovered from the gel for chemical characterization using a combination of liquid chromatography and mass spectrometry. However, this is a costly and laborious technique useful mostly when trying to identify proteins that are completely unknown or for accurate quantification.

The use of antibodies provides a very sensitive tool to identify the presence and relative proportion of specific proteins but the detection of proteins directly in the gel presents several problems due to diffusion. In order to overcome these, proteins can be electrophoretically transferred from the gel to a thin membrane, where they are immobilized. This procedure, known as Western blotting, has been adapted by Towbin et al. (1) from the technique developed by Southern (2) for the transfer of DNA (Table 1).

It should be noted that while for SDS-PAGE electrophoresis standard conditions recommended by equipment manufacturers are quite adequate for most protein extracts, Western blotting can require careful optimization. For proteins of medium to high abundance and not too low molecular weight, almost any condition will work, while proteins of low abundance and/or low or very high molecular weight require much more rigorous empirical testing.

Equipment

A wide variety of electroblotting equipment is commercially available, but there are only two basic designs. In both, the transfer occurs by applying an electric field perpendicular to the plane of the gel. The main difference between them is the cost of the equipment and the time required for the transfer.

Vertical buffer tanks

The polyacrylamide gel containing the separated proteins is placed next to a membrane in a cassette and suspended in a tank containing buffer between two electrodes (Fig. 1).

As the electrodes are several centimetres apart the voltage gradient that can be applied should not exceed 5 V cm–1 to avoid overheating; refrigeration is needed when applying currents of 200 mA or more.

Semi-dry blotting systems

These use buffer-wetted filter paper instead of buffer, in close proximity with flat-plate electrodes (Fig. 2).
In this system, much higher field strengths can be achieved with lower current and cooling is not required. The maximum applied current is 0.8 mA cm–2 and the transfer takes 1–2 hours.

Membranes

The most commonly used membranes for protein transfer are nitrocellulose and polyvinylidene fluoride (PVDF). Both membranes are available with pore sizes of 0.2 and 0.45 µm. The larger pore size is used for most transfers, while the 0.2 µm pore is more efficient for low molecular weight proteins.

Proteins are thought to attach to the membranes by a combination of hydrophobic and electrostatic interaction. The binding capacity of nitrocellulose is 80–100 µg cm2 while PVDF membranes can bind 100–200 µg cm2 and have superior mechanical strength, chemical resistance and protein detection.

As a drawback, PDVF membranes are hydrophobic and will not wet-out in aqueous buffers. They must be wet first in a solution of 50% or more of ethanol, methanol or isopropanol, then rinsed in water and finally equilibrated with buffer.

Transfer buffers

Transfer buffers must be conductive and maintain the solubility of the proteins without interfering with the adsorption of the proteins to the membrane. Traditionally they consist of a buffer of pH higher than the isoelectric point of most of the proteins in the extract, and methanol. The most common formulations are: 25 mM Tris, 192 mM glycine, methanol 10–20% v/v, pH 8.3 and 48 mM Tris, 39 mM glycine, methanol 10–20% v/v, pH 9.2.

Methanol in a concentration of 10 to 20% stabilizes the dimension of the gel and strips SDS from the protein molecules.

Gels swell during the run and this could affect the resolution of the proteins. As swelling depends on polyacrylamide concentration, gradient gels expand unequally, acquiring a trapezoid shape.

On the other hand, proteins of high molecular weight can have limited solubility in the presence of methanol. If the protein of interest is more than 100 kD, the concentration of methanol should be lowered to 5% or even removed completely.

For routine blotting, excess SDS should be removed from the gel by equilibrating it with the transfer buffer for 5–10 minutes. SDS interferes with the ability of the protein to bind to the membrane; this affects particularly low molecular weight proteins. Also, SDS bound to the protein causes it to migrate faster through the membrane, not allowing enough contact time for the protein to adsorb to it. However, when trying to transfer very hydrophobic proteins, like protein from cell membranes, it is worth considering adding a very small amount of SDS (0.05%) as these proteins may precipitate in the gel in the absence of SDS.

Staining

Once the transfer has finished it is important to stain the gel with coomassie brilliant blue to make sure that the transfer has been complete. The fact that your pre-stained molecular weight markers have transferred to the membrane does not necessarily mean that all the proteins in your extract have, particularly the high molecular weight ones. In order to monitor the quality and efficiency of the transfer, the membrane should also be stained. There are several reversible stains that are compatible with immunodetection: Ponceau S red, Fast green FC and 3,4′,4”,4-copper phthalocyanine tetrasulfonic acid and tetrasodium salt (CPTS) are not very sensitive (1–5 mg protein per band) but are very fast and perfectly adequate for checking transfer homogeneity and loading. Spyro Ruby and Spyro Rose (Invitrogen) are highly sensitive fluorescent stains (1–2 ng per band). Reversible membrane protein staining kits of proprietary formulation are available from Thermo Scientific (Pierce) and Invitrogen

Blocking

After the transfer of proteins from the gel to the membrane it is essential to block the unoccupied surface in order to avoid unspecific binding of the antibodies used for protein detection. Blocking can be achieved using a variety of proteins and non-ionic detergent solutions, but no blocking agent is ideal for every occasion, so more than one should be tested to obtain optimum results. The most common blocking agents are bovine serum albumin, non-fat milk, casein, gelatin and dilute solutions of Tween-20 (0.05–0.1%). Buffers used to prepare blocking solutions should have ionic strength and pH as close as possible to physiological conditions; phosphate-buffered (PBS) or Tris-buffered (TBS) saline are the most commonly used.

Using the wrong blocking agent or wrong blocking conditions can obscure the protein of interest; some contain products that cause a high background. For instance, milk contains biotin and glycoproteins; as a consequence neither biotinylated nor lectin-bound antibodies can be used.

Blocking buffers of proprietary formulations are commercially available, like Casein blocking buffer (Sigma and Thermo Scientific), SuperBlock (Pierce), Pierce Protein-Free blocking buffer (Pierce) and ECL blocking agent (GE Healthcare). PhosphoBLOCKER was specifically developed for blocking membranes prior to detection of phosphoproteins by Cell Biolabs, to enhance phosphoprotein signal without increasing background. However, even manufactures show contradictory results when extolling the virtues of their blocking agents, so it is important to determine empirically which one is the best for the protein of interest.

Protein detection

Detection of a specific protein immobilized on a membrane with an antibody is called immunodetection. The antibody that binds to the protein of interest is the primary antibody. Once the primary antibody has been allowed to bind to the target protein, the membrane is washed and incubated with a secondary antibody conjugated to an enzyme that will pinpoint the location of the protein. The secondary antibody is raised against immunoglobulins of the animal species used to raise the primary antibody. Antibodies are diluted in blocking solution to avoid unspecific binding to the membrane. The presence of Tween-20 in the diluent prevents aggregation of the antibodies. It is important that the concentration of Tween-20 does not exceed 0.05% (v/v) as it has the potential to remove a proportion of the protein from the membrane.

The dilutions of primary and secondary antibody must be optimised for each target protein. A non-specific signal can be avoided with higher dilution of the primary antibody; high background can be minimized by higher dilution of the secondary antibody.

Washing of the membrane is necessary to remove unbound antibodies: too little washing will lead to a high background and too much washing can elute the antibodies. Washes are done in PBS or TBS containing 0.02–0.1% Tween-20. The right number and length of washes must be determined experimentally, starting with 3 washes of 5 minutes.

Detection

The most sensitive detection methods are based on an enzymatic reaction, using secondary antibodies bound to enzymes like horseradish peroxidise (HRP) or alkaline phosphatase (AP), β-galactosidase and glucose oxidase. The activity of these enzymes can be detected with chromogenic, chemoluminescent and fluorescent substrates.

In chromogenic detection the enzyme’s reaction results in an insoluble coloured precipitate. WesterBreeze Chromogenic (Invitrogen), Amplified AP ( Bio-Rad) and Immun-Bolt BCIP/NBT (Bio-Rad) are some of the kits commercially available for chromogenic detection. However, this type of detection is not very sensitive, requiring 1–2 ng per protein per band. In chemoluminescent detection (Fig. 3) the enzyme catalyses a reaction that results in the production of light which is detected on a film; the sensitivity is at least 10 times higher than the chromogenic method, making it the method of choice.

Several chemoluminescent detection products are available from Thermo Scientific, Bio Rad and GE Healthcare, all equally suitable. However, there are products that claim to detect proteins in the low femtogram level that are probably very effective, but tend to give high backgrounds and require laborious optimization in order to get the right noise-to-signal ratio. ECL and ECL plus from GE Healthcare give very good signals, adequate for most circumstances. The only drawback of this method is that the signal is not permanent and fades after a few minutes. The reaction can be repeated if needs be, but the background tends to increase.

Fluorescence detection uses a fluorogenic substrate that fluoresces at the site of the enzyme. This method is less sensitive than the chemoluminescent but it has the advantage that the signal is stable indefinitely so blots can be re-imaged. Invitrogen offers two products for fluorescent staining: DyeChrome which has two different fluorescent products, one yellow and one red, which allow the detection of two proteins at the same time and Ampex Gold, which produces bright yellow spots. They can be detected and recorded with a UV epi-illuminator.

Stripping

Once one of the proteins has been identified, the membrane can be stripped and re-probed with a different antibody. However, during stripping some of the proteins on the membrane can be partially removed so it is not always successful.

The most common methods for stripping are: 2% SDS, 100 mM β-mercaptoethanol, 50 mM Tris, pH 6.8. The membrane is incubated at 50ºC for 15–30 minutes and rinsed several times in TBS.

A milder stripping buffer is 0.1 M glycine HCl (pH 2.5–3.0). This buffer will dissociate most antibody–antigen interactions in less than 30 minutes at room temperature or 37ºC.

A simpler and very effective approach is to re-probe the membrane without stripping; two or three antibodies can be used sequentially, starting with the one that gives the cleanest background. Ideally, the proteins to be detected using this method should have relatively different molecular weights but there are instances where two proteins with very similar molecular weights can be detected sequentially providing one of them gives a stronger signal. For instance, connective tissue growth factor (CTGF, MW 38,000) and glyceraldehyde-3-phosphate dehydrogenase (MW 36,000) can be detected in this way as long as the antibody against CTGF, which gives a weaker signal, is used first.

Storage

Membranes can be stored for use at a later date. It is recommended that they are stored dry, between two sheets of Whatman 3MM, protected by cardboard and inside a plastic bag. They can be stored at 4ºC or –20ºC for up to 2 months or at –70ºC for long-term storage. I have successfully re-probed a membrane that had been stuck in my note book, wrapped in cling film for several months. Sometimes it is a matter of luck.

References

Towbin H, Staehelin T & Gordon J (1979). Proc Natl Acad Sci U S A 76, 4350–4354.
Southern EM (1975). J Molec Biol 98, 503–517.