Western blot

Western Blot (WB) is a common method to detect and analyze proteins. It is built on a technique that involves transferring, also known as blotting, proteins separated by electrophoresis from the gel to a membrane where they can be visualized specifically. The procedure was first described by H. Towbin et al in 1979 (Towbin, Staehelin, & Gordon, 1979) and two years later given its name by W. Neal Burnette (Burnette, 1981). Towbin et al described electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets where the original gel pattern was accurately obtained. The setup consists of a standard set of seven steps, Figure 1.

Figure 1. The standard steps in Western Blotting.

Samples are prepared and loaded on to a gel and during the electrophoresis the negatively charged proteins move toward the positively charged anode. In order to further analyze the proteins, they are transferred onto a membrane in a procedure called blotting. After the transfer, the membrane is blocked in order to prevent unwanted membrane-protein interaction in the following steps. To visualize the protein of interest the membrane is commonly first probed using a primary protein-specific antibody followed by a labeled secondary antibody used for detection. An image is taken of the membrane and the result is analyzed.

By adding a separate marker solution to one of the wells in the gel, it is possible to estimate the size of the protein in addition to the antibody interactions that are used to verify the specific protein. The separation on the gel is not only due to size but also to some extent depending on the molecular charge, hydrophobic regions, and degree of denaturation. The setup of the experiment can be varied in many ways to best suit the specific inquiry. When analyzing the results, variations between lanes regarding loading and transfer rates between blots, must be taken into consideration. In addition, the non-linear relation of the generated signal across the concentration range of the samples is also an aspect of consideration when interpreting the results. The outcome of a WB experiment depends on three important factors; the ability of the antibody to bind a specific protein, the strength of the interaction, and the concentration of the protein of interest itself. Moreover, the specificity of the binding to the target and a low cross reactivity are important features as well. The result form the WB is not always easy to interpret as the size of the protein may vary from the theoretical weight due to posttranslational modifications, such as glycosylation, or interactions with other proteins. However, WB is a very common method and almost all available commercial antibodies have been validated using this method.

Technology

Sample preparation

The first step of a WB is to prepare the sample, e.g. tissue, cells, or other solution, which is going to be analyzed. Usually the tissue needs to be broken down by blending, homogenization, or sonication. Buffers are added to lyse the cells and solubilize the proteins and often an inhibitor is added to prevent denaturation or degradation. Different types of filtration and centrifugation methods are applied to further prepare the samples. It is important to determine the total protein concentration of the generated extract to be able to load a specific amount on the gel to enable comparison between samples. Usually a biochemical assay is used in order to determine the protein concentration. The extract is then diluted with loading buffer consisting of glycerol and a dye (e.g. bromophenol blue). The glycerol is used to simplify the loading by raising the density of the extract and the dye is added to visualize the sample. Heat is applied on the samples in order to break the structures of the protein, which will help keeping the negative charge from neutralization (Mahmood & Yang, 2012). Preferably positive and negative controls are included in the set up to confirm identity of the protein as well as the activity of the antibody.

Gel electrophoresis

After sample preparation the extract is ready to be loaded to separate the proteins according to size by gel electrophoresis. An electric field is applied over the gel that causes the charged molecules to move. In WB polyacrylamide gels are used for protein separation and the method is therefore called polyacrylamide gel electrophoresis (PAGE) when using native condition. For denaturing conditions, sodium dodecyl sulfate (SDS) is added to the system and the method is therefore called SDS-PAGE. The SDS binds to the protein and form a negatively charged micelle around the protein regardless of inherent charge. The denaturing condition dissolves the tridimensional structure of the proteins and the charge of the protein becomes relative to its size resulting in separation of the proteins only by size. When using native conditions the mobility is depending on both charge and hydrodynamic size allowing detection of changes in charge due to chemical degradation, conformational changes due to folding or unfolding, aggregation, or other binding events.

The gel typically consists of two sections with different densities: (i) a stacking gel, and (ii) a separating gel, Figure 2. The differences between the two sections are in pH and gel concentration. With somewhat acidic pH and a lower concentration of acrylamide the stacking gel separates the proteins poorly but allows them to form highly defined sharp bands before they enter the separating gel. With more basic conditions and higher gel concentration, the separating gel makes the proteins differentiate by size as smaller proteins travel faster in the gel than bigger ones. Precast gels are convenient; however, it is possible to cast them by hand. The gel is immersed in buffer and the protein samples and markers are loaded to the wells in the gel. A voltage is applied on the gel and the proteins will start to travel down the gel due to their negative electrical charge. Selecting the proper voltage is important since too high voltage will overheat the gel and maybe deform the bands.

Figure 2. A typical gel immersed in buffer.

Blotting to membrane

After gel electrophoresis the proteins are transferred to a solid support membrane, which is the third step of Western Blot. In the transfer process voltage is applied to transfer the proteins from the gel to the membrane. The setup includes sponges, filter papers, the gel, and the membrane, which is placed between the gel and the positive electrode, Figure 3. This ensures the migration of the negatively charge proteins from the gel to the membrane. There are three types of membranes: nitrocellulose, polyvinylidene difluoride (PVDF), and nylon. Even though nylon membranes are superior in several aspects, the high background binding and irreversible staining of some dyes makes this type of membrane less common than the other two alternatives. The major advantage of nitrocellulose membranes is the low background regardless of detection method. Due to a relatively large average pore size, nitrocellulose membranes should not be used for transfer of proteins with low molecular weight. Moreover, when dry, the membrane becomes brittle which makes them difficult to handle. The more stable PVDF membrane allows relabeling and is more convenient to store. The hydrophobic nature of PVDF result in high protein binding capacity, however, as a consequence the background is also higher.

Figure 3. The proteins in the gel are blotted to a membrane and the sample is visualized through blocking, adding antibodies, and washing according to a certain schedule.

There are two methods for the blotting called wet and semi-dry. The wet conditions are preferred when the transfer must be efficient and give high quality regarding distinct and sharp bands. In addition, this is the better choice for transfer of larger protein complex. The gel, membrane, and filter papers are completely immersed in buffer during the transfer and there is no risk of drying out the gel. Semi-dry blotting is more rapid and less volume of buffer is needed. However, this transfer method is usually less efficient, especially for larger proteins, and there is a risk of overheating and drying the gel when using extended transfer times.

Antibody probing

The forth step of the WB is antibody probing. In order to prevent unspecific binding of the antibodies to the membrane, rather than binding specific to the protein of interest, a substance is used to block out the residual sites on the membrane. Common substances used are dried non-fat milk, 5% Bovine Serum Albumin (BSA) diluted in Tris Buffered Saline Tween (TBST), normal goat serum, casein, or fish gelatin (Mahmood & Yang, 2012). Milk is easy to get hold of and inexpensive, however not suitable for all detection labels. Fish gelatin gives lower background but can mask some proteins as well as being a relatively expensive blocking buffer. BSA is inexpensive, whereas serum can contain immunoglobulins giving rise to cross-reactivity. Careful selection of the blocking agent is key since none of the blocking buffers are ideal for all different antigen-antibody interactions. The blocking procedure consists of incubating the membrane in the appropriate blocking buffer for an hour or longer. When using long incubation times, the blocking should be performed at +4°C to rule out the risk of staining artifacts or background. Blocking is a delicate balance between reducing the background without decreasing the signal from the protein of interest.

The blocked membrane is thereafter incubated with the primary antibody. The antibody is diluted to a suitable concentration in TBST, phosphate buffered saline (PBS), or wash buffer. It is preferred to incubate the antibody with BSA if the antibody is going to be re-used. After washing the membrane, the membrane is incubated with the secondary antibody that binds to the primary antibody. The secondary antibody is labeled with a reporter. When using a polyclonal antibody as secondary antibody, it may give rise to some background. In the case of background staining, the secondary antibody may be pre-blocked with non-immune serum from the host it was generated in. Optimization of the concentration of the secondary antibody is recommended due to quite extended variations between antibodies as well as detection system used.

Detection

In the fifth step of a WB, the protein-antibody-antibody complex is detected on the membrane. There are several kinds of labeling of the secondary antibody, e.g. enzymes, fluorophores, biotinylation, gold-conjugation, and radioisotopes, as exemplified in Figure 4. Amongst enzymes the most common is HRP used together with chemiluminescent, chemifluorescent, or chromogenic substances. HRP has a high substrate specificity giving low background, is stable, and inexpensive. In chemiluminescense the HRP enzyme catalyzes the oxidation of luminol from the luminol peroxide detection reagent. The multi-step reaction generates light emission. Certain chemicals like phenols can enhance the emitted light. A direct method is the use of fluorescence; the fluorophores emit light after being excited and no detection agent is needed. It is well suitable for quantitative Western and since different fluorophores emit light of different wavelengths it is possible to perform multiplexing and specific detection of more than one protein at the time. Using a chemical and/or an enzyme to induce the generation of an active fluorophore from a fluorogenic substrate is called chemifluorescence. To further enhance the signal intensity a two-step biotin streptavidin based system may be used. Gold conjugation is also a method where proteins stain dark red due to accumulation of gold. It is also possible to use radioisotopes but they require special handling and are quite expensive.

Figure 4. Different reporter systems.

Imaging

Imaging is the sixth step of WB and the capturing can be analogue using a film, or digitally preformed with a CCD camera or scanner capturing the different kinds of emitted signals. The CCD imaging device enables quantitation with high detection sensitivity and a broad linear range with no chemical waste or need for a dark room. It may be used to detect membranes, stained gels, or for ultraviolet light applications.

Analysis

The last step of a WB is to analyze the results. In a typical qualitative application, the presence of a protein of interest is confirmed, the amount is approximated by visual inspection, and the size is determined by comparison with a marker. Improvements and developments, especially towards highly sensitive detection reagents and advanced imaging techniques, make WB a potential tool for quantitative analysis. The quantitative applications entail a definition of the amount of protein in relative or absolute terms. Some factors are to take under consideration like sensitivity, signal stability, linear dynamic range, normalization, and the signal-to-noise ratio. The minimum of protein that can be seen in a given assay gives the limits of detection (LOD), and the limit of signal intensity that can be reliably used for precise quantification is the limit of quantification (LOQ). Factors that affect these terms are antibody quality and concentrations as well as exposure times when considering the minimum amount of protein detected. A stable signal system expands the time window for reaching high sensitivity, multiple exposures, and possibility to detect weak bands. The range that allows an even and precise quantitation where the signal intensity still is proportional to the amount of protein is called the linear dynamic range. It is important to avoid signal saturation due to excessive amounts of protein or high concentrations of antibodies. A low LOD and quantitation of both weak and strong signals gives a broad linear dynamic range. The protein of interest should be normalized to an internal reference that allows fluctuations in amount of protein loaded onto each well or different concentrations. This can be achieved with housekeeping or spiked protein. The ratio between the signal and noise is important in order to properly quantitate the protein. This is of outmost importance when detecting weak bands where a higher background is expected.

Specific examples

Although the theoretical weight of many proteins differ from the real weight due to post translational modifications, almost all commercial antibodies are validated using WB.

Within the Human Protein Atlas project WB is used for quality control of the polyclonal antibodies generated in the project. After purification, the antibodies are used to detect bands in a setup of lysate and different tissues. The use of siRNA WB is currently implemented to further analyze the antibodies.

The entire WB protocol, including dilution of both primary- and secondary antibodies and the final detection step, is performed in a routine manner and no specific experimental optimization is made for individual antibodies. At first, total protein lysates from selected cell lines and tissues (15?g of RT-4, U-251MG, liver, and tonsil as well as 25?g of HSA- and IgG-depleted plasma) and a marker (PageRuler Plus Prestained Protein Ladder; Thermo Scientific) are loaded on precast 4–20% Criterion SDS–PAGE gradient gels (Bio-Rad Laboratories) and run under reducing conditions, followed by transfer to PVDF membranes (Bio- Rad Laboratories) using Trans-Blot turbo (Bio-Rad Laboratories) according to the manufacturer's recommendations. The Criterion SDS–PAGE gradient gels together with the marker make it possible to analyze protein in the sizes ranging from 10 to 250 kDa. Membranes are then activated in methanol before blocking (5% dry milk, 0.5% Tween 20, 1× TBS; 1 mM tris–HCl, 0.15 M NaCl) for 1 h at room temperature during constant shaking. The membranes are then incubated with primary HPA antibody diluted to ~0,6?g/ml, for 1 h, followed by washing (1 mM tris–HCl, 0.15 M NaCl, 0.05% Tween) and incubation for 45 minutes with the secondary peroxidase-conjugated antibody (swine anti-rabbit 1:4000, Dako). For commercial antibodies a dilution of 1:500 is used and the secondary antibody is either swine anti-rabbit (1:3000, Dako) or goat anti-mouse (1:3000, Dako). A CCD-camera (Bio-Rad Laboratories) is used for detection of signal from the substrate (Immobilon Western Chemiluminescence HRP Substrate; Millipore).

A typical Western Blot from HPA is seen in Figure 5.

Figure 5. Typical Western Blot result using HRP and a CCD camera.

References and Links

The article naming the method and describing it in more detail:
Burnette WN. 1981. "Western blotting": electrophoretic transfer of proteins from sodium dodecyl sulfate--polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. Anal Biochem.
PubMed: 6266278 

The article describes Western Blotting technique, theory, and trouble shooting:
Mahmood T et al, 2012. Western blot: technique, theory, and trouble shooting. N Am J Med Sci.
PubMed: 23050259 DOI: 10.4103/1947-2714.100998

The first article describing electrophoretic transfer of proteins to membrane:
Towbin H et al, 1992. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. 1979. Biotechnology.
PubMed: 1422008 

Western Blotting Principles and Methods - a free handbook provided by GE Healthcare:
http://www.gelifesciences.com/webapp/wcs/stores/servlet/catalog/en/GELifeSciences-se/service-and-support/handbooks

Western Blotting in the Human Atlas Protein project:
http://www.proteinatlas.org/about/assays+annotation#wb

Wikipedia - relevant links:
Polyacrylamide gel electrophoresis

Western blot encyclopedia - A lot of useful information about Western blot as well as troubleshooting, protocols and antibody selections:
http://www.western-blot.us

Antibodypedia - An open-access database of publicly available antibodies and their usefulness in various applications:
http://www.antibodypedia.com

VIDEO: Step by step Western blotting, a tutorial made by Amersham™ ECL™ Prime:
https://www.youtube.com/watch?v=Fozv11nyjzE&feature=relmfu