Factors Influencing Western Blot Results
There are many factors that influence the quality of results observed when performing western blotting. These factors include the membrane used during the transfer, the type of transfer method, and the size of the proteins being transferred. Additionally, the blocking buffer and antibodies used during the probing of the membrane, the detection reagents used, and the probing technique can affect the results.
Another method for transferring the proteins is called electrophoretic blotting (or electroblotting) and uses an electric current to pull proteins from the gel onto the blotting membrane. With either method, the proteins move from within the gel onto the membrane while maintaining the spatial separation they had within the gel. Once transferred to the membrane the proteins are accessible for detection.
A protein’s properties (i.e., charge, hydrophobicity, etc.) affect its ability to bind to membrane surfaces, so finding the optimal membrane may require trying the protein of interest on different membranes. The two most commonly used membrane materials are PVDF and nitrocellulose. Both varieties of membrane are chosen for their nonspecific protein binding properties (i.e., they bind most proteins equally well). Protein binding is based upon hydrophobic interactions, as well as charged interactions between the membrane and protein. Nitrocellulose membranes are less expensive than PVDF, but are far more fragile and do not stand up well to repeated probing. PVDF has a higher overall binding capacity, but also a tendency for higher nonspecific background binding of antibodies.
Researchers, especially those using the electroblotting method, frequently include methanol when transferring proteins onto a nitrocellulose membrane. The inclusion of methanol in the transfer buffer minimizes swelling of the gel due to heat during transfer, and increases the protein binding capacity of a nitrocellulose membrane. However, it also has the effect of reducing the pore size of the gel, which restricts the transfer of some molecules. Methanol also has the effect of removing sodium dodecyl sulfate (SDS) from the proteins, which can also inhibit transfer and may favor renaturation of some proteins. If methanol is left out of the transfer buffer, it is important to pre-equilibrate the gel in transfer buffer for at least 30 minutes prior to electroblotting.
One of the key steps in the western blot workflow is the transfer of proteins from the polyacrylamide gel after electrophoresis to the nitrocellulose or polyvinylidene difluoride (PVDF) membrane so that specific proteins can be detected using immunodetection techniques. As previously discussed, methods for achieving this protein transfer include capillary transfer and electrophoretic transfer. Life Technologies has electrophoretic transfer systems using wet, semi-dry, and dry methods outlined below.
The key difference between these systems is the amount of buffer used during the setup of the transfer sandwich. In traditional wet and semi-wet transfer systems, the membrane-gel sandwich is submerged into a tank that contains transfer buffer. A current is passed through the buffer to move proteins from the gel onto the membrane. For semi-dry transfer, the membrane-gel sandwich is flanked by filter paper soaked with blotting buffer. Charge is driven through the filter paper to move the proteins from the gel to the membrane. In dry transfer systems, the membrane-gel sandwich is placed between gel matrices that contain ions. These ions move when current is applied, resulting in transfer of the proteins from the separation gel to the membrane.
Transfer efficiency is influenced by molecular weight; proteins smaller than 60 kDa are transferred more efficiently than larger ones, irrespective of transfer buffer. The addition of 0.1% SDS has been reported to improve transfer of larger proteins, but may cause a general reduction in protein binding to a nitrocellulose membrane, due to interruption of hydrophobic interactions. In addition, the presence of detergent and the heating caused by high transfer current may have adverse effects on epitope stability, and consequently adversely affect the antibody-based detection of transferred proteins.
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The uniformity and overall effectiveness of the transfer of protein from the gel to the membrane can be checked by staining the membrane with a reversible dye, such as the Novex® Reversible Membrane Stain.
The reversible nature of this stain allows you to subsequently destain the membrane without affecting downstream probing of the membrane with antibodies.
Because membrane materials are selected for their ability to bind protein, and both antibodies and their targets are proteins, steps must be taken to prevent interactions between the membrane and the antibody used for detection of the target protein. Blocking of nonspecific binding is achieved after transfer by placing the transfer membrane in a blocking solution such as WesternBreeze® Blocker. The blocking solution binds to any part of the membrane where transferred proteins have not already attached. Then, when the membrane is probed with antibody, the nonspecific protein-binding sites are already occupied and the antibody binds only to the specific target protein. This reduces “noise” in the final product of the western blot, enabling clearer results and minimizing false positives.
Antibodies are critical to the success of the western blot technique. They allow for the selective detection of the protein of interest amid a vast array of other proteins. Typically a primary antibody is used to specifically bind the protein of interest and a labeled secondary antibody is used for detection. The primary antibody you choose for western blot needs to bind to a denatured form of the target protein, usually to a unique portion of its primary amino acid sequence. An antibody that effectively binds a protein on the surface of a cell may work very well for flow cytometry because it recognizes the protein’s folded, native structure, but fail to detect it on a western blot for the same reason. However, western blotting is the most common application for antibodies, so commercially available primary antibodies are often produced to detect proteins on western blots (and then tested and certified for western blotting applications).
Primary antibodies are typically diluted from their stock concentration prior to use, and each antibody requires some optimization in order to perform at its best. The typical dilution range for a Life Technologies primary antibody used for a western blot is 1:500 to 1:5,000 (check the antibody manual; some antibodies may require more or less dilution). After the blot is incubated with the primary antibody solution, the blot is washed, and if the primary antibody is not labeled with a detection molecule, a secondary antibody or other secondary detection reagent is added to the blot.
The secondary antibody can be conjugated to a number of different molecules for detection, such as enzymes, fluorophores, dyes, and haptens for signal amplification. The most common means of detection is to use a secondary antibody conjugated to horseradish peroxidase (HRP) or alkaline phosphatase (AP) enzyme. Recently, however, there has been a move toward using fluorescently labeled secondary antibodies that can be imaged on a scanner designed to detect fluorescence. Fluorescence-based detection provides sensitivity similar to that of chemiluminescence detection but allows for the detection of multiple fluorophores at the same time, to give comparative data for two or more different proteins.
The use of secondary antibodies can greatly increase sensitivity compared to the use of a labeled primary antibody. Directly conjugated primary antibodies usually have a relatively small number of labels conjugated per antibody. Secondary antibodies are designed to bind the primary antibody in more than one place, which results in several secondary antibodies being bound to the primary antibody, each with their own labels or enzymes. The resulting 3- to 5-fold increase in the number of labels or enzyme present results in a significant amplification in signal. Further amplification can be achieved by using a biotinylated secondary antibody followed by incubation with streptavidin HRP. Although amplification strategies allow you to detect low-abundance targets, they also introduce new variables. Each reagent needs to be titrated to determine the concentrations which provide the best signal-to-noise ratio.
Colorimetric detection depends on incubation of the western blot with a substrate for a reporter enzyme that is bound to the primary or a secondary antibody used to probe the blot. The most frequently used reporter enzymes are horseradish peroxidase and alkaline phosphatase. The enzymatic reaction converts the soluble substrate dye into an insoluble form of a different color that precipitates next to the enzyme and thereby stains the membrane. Development of the blot is then stopped by washing away the soluble dye. Protein levels are evaluated for stain intensity using densitometry or spectrophotometry. Colorimetric detection is simpler, but less sensitive than other methods. Also, the colored precipitate generally cannot be removed, so these membranes cannot be stripped for reprobing.
Chemiluminescence detection depends on incubation of the western blot with a substrate that will luminesce when exposed to the reporter enzyme that is bound to the primary or secondary antibody. The light is then detected either by photographic film, or by CCD cameras or scanners, which capture a digital image of the western blot. The image is analyzed by densitometry, which evaluates the relative amount of protein staining and quantifies the results in terms of optical density of the stained image. Newer software allows further data analysis such as molecular weight analysis, if appropriate standards are used.
A fluorescent label on a probe is excited by light, and the emission of light energy from the dye is then detected by a photosensor, such as a CCD camera equipped with appropriate filters. The camera can capture a digital image of the western blot, which can be further analyzed to gain information such as molecular weight and semiquantitative western blot data. Fluorescence is gaining popularity and, when used optimally, can be considered among the most sensitive detection methods for blotting analysis.
Western blotting of proteins is an essential part of many workflows because of the high sensitivity and specificity that can be obtained. However, the manual process is long and time-consuming. A typical western blotting protocol can take between 6 and 16 hours to perform, and a significant part of the time is spent on processing the western blot for detection. The western blot processing may include more than a dozen individual blocking, washing, incubation, and rinsing steps. This lengthy and tedious process can result in inconsistencies and errors. The BenchPro® 4100 Western Processing System is designed to eliminate the need for manual processing of routine liquid-handling steps. With an on-board CPU and an intuitive interface, the BenchPro® system makes it easy to create and run western protocols reproducibly and with minimal errors.