A successful Western blot requires considerable time, effort and the correct reagents. Careful attention to detail is the key to a great result. These five tips can help eliminate pitfalls that can wreck the product of your hard work.
#1. Optimize Your Blocking Buffer
There are many different sources and types of blocking reagents sold for Western blot applications. Antibody performance can be compromised by choosing a poor blocking reagent. Milk-based blockers may contain IgG that can cross-react with anti-goat antibodies. This can significantly increase background and reduce sensitivity. Milk-based blockers may also contain endogenous biotin or phospho-epitopes that can cause high background noise. BSA is a good alternative for phospho-protein detection, detection using Alkaline phosphatase and for avidin/biotin detection systems. For those doing chemiluminscence detection it is essential to avoid blocking buffers containing azide, which can interfere with the HRP enzyme reaction. Nonmammalian-based blockers as well as nonprotein blocking agents are also available and can be preferable in some situations. Optimization using several blocking buffers is essential to determining the best blocker for your Western blot protocol.
The addition of detergents to diluted antibodies can significantly reduce the background on the Western blot. Optimal detergent concentration will vary, depending on the antibodies, membrane type, and blocking reagent. Keep in mind that primary antibodies have different binding affinities and may be washed away by the addition of too much detergent. Never expose the membrane to any detergent until blocking is complete, as this may cause an increase in background. Primary and secondary antibody diluents should have a final concentration of 0.1–0.2% Tween®20 for nitrocellulose membranes, and a final concentration of 0.1% for PVDF membranes. Higher concentrations of Tween 20 may increase background on PVDF. When using PVDF membranes for fluorescent detection, secondary antibody diluents should have a final concentration of 0.01–0.02% SDS. SDS can also be added to the antibody diluents when using nitrocellulose to dramatically reduce the overall background and to reduce or eliminate nonspecific binding. It is critical to know that SDS is an ionic detergent and can disrupt antigen-antibody interactions when too high a concentration is used at any time during the detection process.
#3. Primary Antibody
An antibody that binds directly to the molecule of interest and is produced to detect a specific antigen is called the primary antibody. Primary antibodies can be produced in a wide variety of species as either monoclonal antibodies in mice and rabbits or as polyclonal antibodies in mice, rabbits, goats, rats, guinea pigs, chickens, humans, and many others. Different primary antibodies that are produced to detect the same antigen can actually perform very differently. It may be necessary to test multiple primary antibodies, optimizing for the best concentration to use to achieve the best performance in your Western blot system. Reuse of primary antibody solutions is not recommended; the extent of antibody depletion is unknown and the storage of diluted antibody in detergents can result in inactivation of the antibody and poor results. Repeated freeze/thaw of antibodies and dilute antibody solutions can also damage the antibody molecules.
#4. Secondary Antibody
There are many choices in secondary antibodies for Western blot detection such as whole IgG, subclass specific, and heavy and light chain specific to name a few. The choice depends on the primary antibody used. For two-color fluorescent detection, always use highly cross-absorbed secondary antibodies. Failure to do so may result in increased cross reactivity. The concentration of secondary antibody used for Western blots can vary greatly, and should be optimized for your Western blot system using the manufacturer’s suggestions for each individual application. In chemiluminescence detection, reduction of the secondary antibody concentration can significantly reduce the amount of background. Optimizing your antibody concentrations ultimately saves time, reagents, and headaches.
#5. Keep It Clean
Your Western blot results can be greatly affected by your attention to detail. Make sure that your incubation boxes and forceps are thoroughly cleaned, rinsed, and dried before you start. Handle membranes only with forceps to prevent fingerprints that are easily seen with fluorescent detection. Do not pour your reagents directly on your blots; instead, pour down the sides of the incubation box or container. Write on blots with pencil, not ink, to prevent unwanted smears and spots. When imaging, be sure your imaging surface is clean and lint free to prevent speckling, smearing, and contamination by other dyes (e.g., Coomassie blue, fluorescent dyes, or residual substrates). Prevent introducing background with chemiluminescence detection by wicking off excess substrate to prevent pooling on the membrane.