The NativePure™ Mammalian Affinity Purification Kit provides an easy-to-use, efficient method for purifying native protein complexes.
NativePure™ pcDNA™ Gateway® vectors (Figure 1) are designed to allow expression of biotinylated recombinant fusion proteins in mammalian cells through N- or C- terminal fusion of your recombinant protein of interest to the capTEV™ Tag.
The capTEV™ Tag consists of a BioEase™ in vivo biotinylation peptide, two Tobacco Etch Virus (TEV) protease recognition sites, and a 6XHis tag. The BioEase™ tag, a 72-amino acid sequence from K. pneumoniae, directs in vivo biotinylation of a specific lysine residue.
The strong interaction of biotin and streptavidin is used to purify the protein complexes under native conditions using Streptavidin Agarose included in the NativePure Affinity Purification Kit. Biotinylated proteins and protein complexes remain bound, while impurities are removed by thorough washing of the Streptavidin Agarose column.
Two tandem AcTEV™ Protease recognition sites facilitate the release of bound biotinylated proteins and protein complexes from Streptavidin Agarose, creating a useful strategy for protein purification.
Following purification, the protein complexes are ready for analysis in many downstream applications including SDS-PAGE, western analysis, mass spectrometry, and native electrophoresis using the NativePAGE™ Novex® Bis-Tris Gel System.
The NativePure™ Mammalian Affinity Purification Kit provides:
• Gateway® vectors designed to express your protein of interest in multiple expression systems
• pcDNA™ vectors with the capTEV™ Tag that allow in vivo biotinylation of your protein of interest
• Streamlined purification protocols with Streptavidin Agarose
Contents and Storage:
The NativePure™ Mammalian Affinity Purification Kit includes the NativePure™ pcDNA™ Gateway® Vector Kit and the NativePure™ AffinPurification Kit. The NativePure™ pcDNA™ Gateway® Vector Kit and NativePure™ Affinity Purification Kit are also available separately. The NativePure™ pcDNA™ Gateway® Vector Kit consists of the pcDNA™3.2/capTEV™-NT/V5-DEST vector (N-terminal purification tag), the pcDNA™3.2/capTEV™-CT/V5-DEST vector (C-terminal purification tag), and the pcDNA™3.2/capTEV™-NT/V5-GW/ARPC2 control vector. The NativePure™ Affinity Purification Kit contains Streptavidin Agarose, NativePure™ 5X Lysis/Binding Buffer, NativePure™ 10X TEV Cleavage Buffer, 100 mM DTT (dithiothreitol), 10% NP40, AcTEV™ Protease, NativePure™ Columns, and NativePure™ Concentrators. Store Streptavidin Agarose, 10% NP40, NativePure™ 5X Lysis/Binding Buffer, NativePure™ 10X TEV Cleavage Buffer, NativePure™ Columns, and NativePure™ Concentrators at +4°C. Store the NativePure™ pcDNA™ Gateway® Vector Kit, AcTEV™ Protease, and 100 mM DTT at -20°C. All components are guaranteed stable for 6 months when properly stored.