One of the most commonly used molecular biology tools, the Western Blot, known also as an immunoblot, allows for investigation of proteins separated by molecular weight and subsequent detection by cognate antibodies. The Western Blot requires diligent care in processing samples, preparing and using reagents, and interpreting results. Each of these will be discussed below, but in order to understand the best practices for optimizing a Western Blot, the key principles of the technique should be thoroughly understood.
Much like the technology that immediately preceded it, the Western Blot uses principles similar to that of the Northern and Southern blot. All three methods employ electrophoresis to separate samples which are then transferred to a stable membrane and subsequently probed. Incidentally, "Western" and "Northern" are somewhat misnomers, merely following the nomenclature of the Southern Blot, named after its inventor Edwin Southern.
Specifically in a Western Blot, samples of interest are loaded into a well of polyacrylamide gel (PAG) containing sodium dodecyl sulfate (SDS), to which an electric field (E) is applied, hence "SDS-PAGE". SDS both denatures folded proteins, allowing them to travel more easily through the gel, as well as impart a negative charge to them. These negatively charged, linearized proteins then migrate away from the cathode, the negatively charged area near the loading region of the gel, and towards the anode. As they travel, proteins are restricted by pores whose size is determined by the polyacrylamide and cross-linker percentage of the gel. Higher polyacrylamide percentage yields decreased pore sizes while increasing cross-linker percentage yields larger pore sizes. Larger proteins are retarded and remain closer to the loading wells at the top of the gel while smaller proteins travel further as the migration front, potentially off the gel if proteins are small and run long enough. Once the proteins in the sample have been sufficiently separated by their molecular weight, they are then ready for transfer. Moving the resolved proteins sample to nitrocellulose provides a durable substrate which may then be probed. Immunoglobulins (IgG), usually IgG antibodies are used to specifically detect the protein(s) of interest, hence Western Blots also being referred to as "immunoblots". Antibodies may be directly detected with a signaling molecule (chromogenic, chemiluminescent, or fluorescent), or indirectly detected with a labeled secondary antibody.
While this technical note will speak to the aspects of antibody troubleshooting, there are many features to a Western Blot which can be modified and optimized as outlined in the chart below.
|Low/no detectable signal
|Not enough antigen
|Not enough antibody binding
|Antibody or antigen interaction with blocking buffer
|Inefficient membrane transfer (confirmed with Ponceau S)
|Too much antibody binding
|Binding kinetics too high
|Blocking agent interference or cross reactivity (confirmed by secondary only control)
|Multiple positive bands
|Too much antibody binding
|Antibody binding to blocking/binding buffer precipitate
|Non-uniform spotting, irregular detection across membrane
|Uneven exposure to antibody
Antibodies raised against protein, rather than peptide antigens, present the immune system with conformationally relevant epitopes. This is critical when performing a native, or non-reducing Western Blot, but serves reducing Western Blots as well. As reducing conditions denature proteins, some epitopes maybe lost due to conformational change as the protein is linearized. To insure against this, both polyclonal and monoclonal antibodies intended for Western Blots should be raised against protein antigens. Peptide antigens both increase risk of failing to provide enough non-conformational dependent epitopes and may not recapitulate the structure in its reduced form. However, using a protein antigen is not sufficient to guarantee a Western Blot functional mAb. mAbs should be qualified via Western Blot during the cloning process to appropriately screen for application appropriate positive clones. Additionally, protein epitopes simply afford more epitopes for antibody recognition which may result in more robust signal with a pAb, or greater potential for positive mAb clones.
Just as is the case with an ELISA, polyclonal antibodies have the potential to return greater signal through binding of multiple epitopes. This however is a double edged sword as the complexity of a pAb may also confer a higher degree of non-specificity, introducing noise into the system. Monoclonal antibodies benefit from specificity, but lack the potential signal amplification through binding of multiple epitopes like that of pAbs. If available, amplification may be achieved through mixing of multiple mAbs so long as they recognize unique epitopes.
Raw pAb anti-serum or mAb supernatants can work to detect proteins in a Western Blot, but the complexity of unprocessed reagents may add difficulty to detection. Controlling blot incubation with pre-immune serum or blocking with bovine serum albumin (BSA) may ameliorate noise issues, but antibody purification (minimally with protein A and preferably with antigen affinity) will offer the best results. Should cross-reactivity still be present, or affinity purification unavailable, pre-adsorption is another option to remove non-specific binding. Passing the antibody reagent through a column containing irrelevant proteins, such as BSA, will remove the non-specific antibodies.
Also a consideration for FACS, IHC, and ELISAs, direct detection allows for a simple and fast protocol with potentially cleaner signal to noise ratio. Direct detection utilizes a single antibody for delivery of the signaling molecule whereas indirect detection delivers the signal with a secondary antibody directed against the primary. Though indirect detection may potentially suffer from cross-reactivity, it can have its benefits. Some of which include signal amplification through binding of multiple pAbs to the primary, flexibility to optimize with alternative signal agents, and cost savings through use of a few labeled secondary antibodies for use with many non-labeled primary antibodies.
Signal selection may often be restricted to the equipment available as each requires their own signal specific apparatus for measurement. Beyond equipment access, each means of signal detection via antibody labeling has its own benefits. Chromogenic, also known as colorimetric, and chemiluminescent signal development utilize enzymatic development of color or light respectively. Enzymatic development can provide a robust signal but should be empirically optimized and tested in order to establish a uniform and appropriate developing time. This avoids overdeveloping both the signal of interest and non-specific noise. Fluorescent detection lacks the ability to magnify signal to the degree of enzymatically driven systems, but offers capability of simultaneous multi-protein detection via differing fluorophore labeled antibodies without stripping and re-probing of the blot.
Likely one of the most variable aspects of Western Blotting is the concentration of antibody during incubation. Working anti-serum pAb concentrations can range from 1:100 to 1:500,000 while purified antibody working dilutions typically range from 1 µg/ml to 0.01 µg/ml. Empirical testing using matched loading amounts of protein and serial dilutions of antibody will determine the best concentration. This may require concomitant testing in a matrix with developing time if using enzymatic signal development.
When encountering difficulties outside of the aforementioned considerations, one must investigate less common issues such as potential confounding interactions regarding antigen-antibody binding. Buffers, although commonly designed to include irrelevant proteins (BSA, non-fat milk, etc) and detergents (tween-20, NP-40, etc) may interfere with antibody recognition. Titration, substitution, or omission may be required to achieve sufficient signal. Every component of binding and blocking buffers must be considered. Even common preservatives such as sodium azide (NaN3) may interfere with peroxidase driven detection systems.
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