OriGene Technologies
Sr. Marketing Director
Western blotting is a powerful and commonly used tool for separating and detecting specific proteins from mixtures such as cell lysates, tissue homogenates or immunoprecipitation experiments. They are so integral to protein-based research that it can be frustrating when they don’t work as expected.
What do you do when you reach the end of your Western-blot protocol only to find that the lanes on the transfer filter are completely blank? There is not one easy solution, because there are many steps in the process that that could have malfunctioned. For example, did the transfer step not work properly? Was there something wrong with the antibody? Or did your transfected protein not express to begin with? Here are some tips to help you avoid such Western-blotting headaches.
Overexpressing proteins with cell lysates
Including a positive control lane in your Western blot can help you to understand what worked, and what might have gone wrong. If all the steps in the Western blotting procedure are functioning as intended, then at the very least the positive control lane should show a positive result. If the positive control lane is blank, then you know right away that one or more steps in the blotting process malfunctioned, or that you have a problem with antibody quality (see below).
An excellent source of positive controls for Western blotting is lysate from cell lines that overexpress the gene encoding your protein of interest. Loading such lysates into your positive control lane results in a reliably detectable protein sample. A positive result in the positive control lane can bring you peace of mind. It means all the steps of your Western blot functioned properly, including gel electrophoresis, protein transfer to blotting membrane, membrane blocking and antibody labeling. It also gives you greater confidence that the results in the other lanes are real rather than artifactual.
Detecting low-abundance proteins
Proteins that are rarely expressed or expressed at very low levels can make interpreting a Western blot difficult. For example, a blank Western-blotting filter could indicate a malfunctioning Western-blotting procedure. On the other hand, it could result from protein levels that are simply below the limit of detection.
When trying to detect low-abundance proteins, it is especially important to know that your Western blot is functioning as expected. This is another instance in which overexpression lysates are useful as tools for positive controls. If you detect your protein of interest in the control lane, then an absence of the protein in other lanes is probably directly related to its low abundance rather than a faulty step in the blotting protocol.
Rank your antibodies
Though it might seem that you can find an antibody to almost any protein today, some are not of a quality useful for Western blotting. A high-quality antibody is sensitive and specific for its target, with minimal background detection and cross-reactivity. But unfortunately, not all antibodies are created equal. Some antibodies, for example, are created using shorter peptides—rather than the entire protein—as the antigen. Often the result is an antibody that isn’t as specific as it could be.
Low-quality antibodies can complicate interpretation of Western blots whose readouts depend entirely on properly functioning antibodies. In fact, researchers return up to 30% of antibodies because in practice, the antibodies don’t recognize their target antigens. So it is wise to verify the quality of the antibody you plan to use for Western blotting—before investing a lot of time and money into Westerns whose results depend upon it. Antibody quality control is possible using lysate from cells overexpressing your protein of interest. Running serial dilutions of the overexpression lysate in different lanes of a Western blot can help you assess the antibody’s characteristics. This procedure is also helpful in comparing batch-to-batch differences of the same antibody.
Use a tagged positive control
For running a positive Western-blot control, you need a source of concentrated, specific protein. Lysates from cells that are overexpressing your protein of interest fit the bill perfectly. But one additional feature can make your positive control tool even more valuable: molecular tags on the overexpressed protein of interest. Molecular tags such as -myc or -FLAG tags lend greater ease and reliability to isolating and detecting the overexpressed target protein.
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