A monoclonal antibody was developed that reacts only to the AviTag when fused to the C-terminus of desired protein. It will identify C-terminus fusions on western blots and has been immobilized on agarose beads for purifications purposes. The antibody will recognize biotinylated as well as unbiotinylated AviTag.
AbC antibody—Anti C-terminus AviTag MAB
Purified monoclonal IgG is provided in PBS at 1mg/ml and can be used as any primary antibody in ELISAs, western blots, or any immunological procedure. Monoclonal IgG is purified using an affinity column of immobilized AviTag peptide.
Western blot protocol
Specification sheet for AbC antibody
AbCA agarose- Anti-C-term AviTag MAB Agarose
Purified anti-C-terminus AviTag MAB was conjugated to agarose to create an affinity column for C-terminal fused Avitag protein. Cell extracts generated by common methods can be used as long as reducing agents are NOT USED. High salt concentrations and most ionic detergents do not inhibit the binding capacity of the resin.
After washing the unwanted proteins from the column, the bound protein can be eluted using any typical method of eluting antigen-antibody complexes (pH change etc) If the protein of interest is sensitive to pH, we suggest the mild method of elution used by Thompson et.al for affinity purifying RNA polymerase (Thompson NE, Hager, DA and Burgess, RR (1992) Biochemistry 31, 7003-7008; Thompson NE and Burgess, RR (1994) Protein Expression and Purification 5,468-475) The resin is regenerated by placing resin in PBS. Resin can be used multiple times (greater than 15 elutions)
Two mls of resin are provided with each order. This will purify on average 1mg of Avitag protein.
BIS-300-Positive Control Substrate Determination of extent of biotinylation
BIS-300 kit contains fully biotinylated and fully unbiotinylated Maltose Binding Protein with Avitag (MBP-AviTag) at the C-terminus. They are used to generate standard curves in a modified ELISA protocol. The extent of biotinylation of the test protein is measured by comparison with known quantities of fully biotinylated MBP-AviTag protein.
The standard protein and unknown proteins are adsorbed to the wells of a 96-well plate at known protein quantities. The biotin associated with the AviTag is detected by its interaction with streptavidin-conjugated alkaline phophatase. Extraneous biotin is removed when the samples are washed after adsorption, eliminating exhaustive dialysis steps. As little as 1ng of biotinylated protein can be detected (Contrary to HABA based assays) This assay is dependent on the ability of the test proteins to bind to the plastic of the 96-well plate. The fully biotinylated and fully unbiotinylated Maltose Binding Protein with Avitag at the C-terminus kit can also be used as control samples for western blots or other applications.