Azure BiosystemsPost-translational modifications occur when proteins are synthesized and many proteins are modified by the covalent addition of chemical groups or compounds to amino acid side chains.
These post-translational modifications change or regulate protein activity, localization, stability, and interaction with other proteins. Many modifications have been described, including phosphorylation, glycosylation, lipidation, and ubiquitination.
Some post-translational modifications, such as ubiquitination, change the migration of the protein on a gel, making it possible to detect the modified protein on a Western blot by the appearance of a new, higher molecular weight band or bands. If the difference in migration is large enough, the modified and unmodified versions of the protein may be analyzed at once using a chemiluminescent Western blot detected with an antibody that recognizes both protein versions.
Other modifications, such as phosphorylation, have little or no effect on protein migration. To analyze phosphorylation by Western blot, two antibodies, one specific for the unmodified version and one for the modified version of the protein must be used. Because the two bands cannot be resolved based on migration, if they are detected using chemiluminescence, two experiments must be done.
Either duplicate blots must be run, each probed with a different antibody, or a single blot can be first probed with an antibody directed to the phosphorylated version of the protein, followed by stripping and re-probing the blot with an antibody specific for the unphosphorylated version. Duplicate blots require using twice as much sample and introduce the potential for inter-experimental variation due to differences in protein loading or transfer efficiency.
However, stripping and re-probing a blot can affect data quality, potentially stripping target protein and reducing the ability to obtain quantitative data about the two protein versions. Additionally, stripping and re-probing is time consuming.
Multiplex fluorescent detection is the best option for analyzing two or more proteins of similar molecular weight by Western blot. Antibodies that recognize different epitopes, such as the phosphorylated and unphosphorylated versions of a protein, can be conjugated to fluorophores with non-overlapping excitation and emission spectra and imaged simultaneously.
Multiplex fluorescent detection allows the most powerful approach to the study of post-translational modifications by Western blot. Detecting both proteins on the same blot improves data quality, removing potential inter-experiment variation. Multiplex detection saves sample and streamlines workflow compared to running duplicate blots.
To enable quantitative analysis of two targets on one multiplex blot, a loading control or total-protein stain detected in a third channel can be included in the experimental design. Three-color Western blotting is possible with the Azure 400 or 600 system, as well as four-color Western blotting with the new Sapphire FL Biomolecular Imager. The Sapphire FL scans with up to three optical modules at once to create a four-channel fluorescent image to allow you to detect up to four proteins on the same Western blot.
Azure also provides fluorescent secondary antibodies, fluorescent Western blot buffers, opaque incubation trays, and other products that can help you on your way to getting the best multiplexing data.
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Editor’s note: since the original publishing date of this article, AzureSpot software has been updated to AzureSpot Pro. Learn more about AzureSpot Pro here. Try a free 2-week
Western blotting is an important tool for researchers studying signal transduction and other processes where understanding the phosphorylation state of a protein is important for
