The TAP (tandem affinity purification) method is an affinity purification method for the isolation of TAP-tagged proteins along with associated proteins. The TAP tag consists of a calmodulin binding peptide (CPB), a tobacco etch virus (TEV) protease cleavage site, and Protein A. However, additional tag combinations have been used with the TAP method including the combination of FLAG tags and HA tags. The TAP method permits identification of proteins interacting with a particular target protein without any prior knowledge about the function, activity, or composition of the interacting proteins. The TAP tag has been especially useful and deployed with yeast TAP-tagged ORF clones. These clones contain genomic fusions of the TAP construct and are extremely useful for determining natural protein interactions and expression level variations based on physiological changes. Invitrogen TAP tag antibodies are designed to reliably detect TAP in various applications.
Invitrogen anti-TAP Tag antibodies are designed to dependably detect TAP tag in various applications.
Western blot analysis of the TAP Tag. Western blot was performed by loading the indicated amounts of a TAP Tag containing control protein and 5 µl of Lane Marker Reducing Sample Buffer (Cat. No. 39000) per well onto a 4-20% Tris-HCl polyacrylamide gel. Proteins were transferred to a PVDF membrane and blocked with 5% BSA/TBST for at least 1 hour. The membrane was probed with a TAP Tag monoclonal antibody (Cat. No. MA1-108) at a dilution of 1:1000 overnight at 4°C on a rocking platform, washed in TBS-0.1% Tween-20, and probed with a goat anti-mouse IgG-HRP secondary antibody (Cat. No. 32430) at a dilution of 1:15,000 for at least 1 hour. Chemiluminescent detection was performed using SuperSignal West Dura (Cat. No. 34075).
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