A 2014 workshop held by the National Institutes of Health, Nature Publishing Group, and Science recommended that best practice guidelines be established for western blot. As a result, publishers have become stricter when reviewing western blot data. Here, we share tips for producing publication-worthy western blots, based on the main steps of a standard workflow.
Unless the target of interest is a secreted protein, which can be detected in culture medium, samples must be lysed in an appropriate buffer for the target location. NP-40 buffer is commonly used for preparing whole cell lysates and extracting membrane proteins, while RIPA buffer is preferred for nuclear and mitochondrial targets.
Lysis buffers should always contain protease inhibitors (and phosphatase inhibitors when detecting phosphoproteins) and be matched to the isoelectric point of the protein analyte (typically pH 6–8). Other best practice recommendations include performing sample preparation on ice, centrifuging lysates to remove cellular debris and chromosomal DNA, and determining the total protein concentration prior to loading.
When detecting native proteins, both the lysis buffer and the loading buffer should be non-denaturing (free from SDS and reducing agents).
Polyacrylamide gel electrophoresis (PAGE) performs best when the correct percent acrylamide is selected for the target analyte. 15% acrylamide is generally recommended for proteins with a molecular weight of 10–50 kDa, 12% for proteins of 40–100 kDa, and 10% for proteins >70 kDa. Gradient gels allow proteins with a range of molecular weights to be resolved in the same assay. For larger proteins (700–4,200 kDa), agarose gels provide better separation. For reliable results, researchers should ensure that the pH of the running buffer is above the protein’s isoelectric point (to maintain a negative charge), and should include positive and negative control samples as well as a molecular weight marker.
The choice of membrane is a key consideration for protein transfer. Nitrocellulose offers a low background signal for most detection methods, although it can become brittle upon drying, presenting challenges for stripping and re-probing. Polyvinylidene fluoride (PVDF) membranes are more robust, but are prone to autofluorescence. Pore size should be observed if the protein of interest is particularly large or small. Other factors to bear in mind include the pH of the running buffer, which should be above the protein’s isoelectric point, and the voltage, which should be optimized to ensure a complete transfer and avoid melting the gel.
Blocking is critical to prevent non-specific antibody binding to membranes. Bovine serum albumin (BSA) and non-fat-dried milk are widely used for blocking as they are inexpensive and readily available. However, BSA should be avoided when using secondary antibodies directed against bovine or related species (e.g., alpaca, goat, horse, or sheep) as it often contains bovine IgG—although it is worth noting that IgG-free BSA is now available. Milk should not be used when detecting phosphoproteins as it contains casein that can bind anti-phosphoprotein antibodies. Ideally, normal serum from the host species of the secondary antibody should be chosen for blocking. This must always be prepared as a fresh 5% w/v solution in Tris-buffered saline (TBS) or phosphate-buffered saline (PBS), of which TBS is recommended when detecting phosphoproteins. The inclusion of 0.1% Tween-20 in blocking solutions will help prevent protein aggregation that could cause unwanted background signal.
There are numerous considerations when selecting antibodies for western blot. Primary antibodies should be supplied with validation data confirming specificity for the target antigen, and should ideally be raised in a different species than the sample to circumvent secondary antibody binding to sample immunoglobulins. When running native western blots, it is important to choose primary antibodies that are raised against the purified protein as they may not recognize their binding epitopes when presented in a linearized form.
Secondary antibodies must be matched to the host species of primary antibodies. If multiple secondary antibodies are to be combined, they should share the same host species to minimize cross-reactivities. If this is not possible, using secondary antibodies that are cross-adsorbed against the host species of the other secondary antibodies in the experiment is advised. When detecting proteins on western blots after immunoprecipitation, anti-light chain and anti-Fc specific secondary antibodies will respectively avoid interference in the 50 kDa and 25 kDa range.
For both primary and secondary antibodies, titrations should be performed to determine the optimal working concentrations, using a suitable diluent to preserve antibody activity. Washing after blocking and between antibody incubations is critical to improve assay sensitivity and should involve submerging the membrane in TBS or PBS containing a low concentration of detergent for 5–10 minutes, with constant agitation.
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