Product. Ni-NTA His Bind Resin. Ni-NTA His Bind Superflow. His Bind Resin. His Bind Magnetic Agarose Beads. His Bind Column. His Bind Quick Resin

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1 Novagen offers a large variety of affinity supports and kits for the purification of recombinant proteins containing popular peptide fusion tags, including His Tag, GST Tag, S Tag and T7 Tag sequences. Different resin formats enable small to large scale applications, including medium pressure purification and high-throughput magnetic separations. All resins and buffer kits are compatible with the BugBuster and CytoBuster protein extraction reagents and Benzonase Nuclease. For proteins expressed as inclusion bodies, we offer the Protein Refolding Kit, which enables simple solubilization and refolding under defined conditions. The compatible Non-Detergent Sulfobetaines (NDSB), as well as other denaturants and reducing agents, can aid in protein refolding and are also useful in other proteomic applications. His Bind Pre-Packed Column HIS BIND PURIFICATION PRODUCTS The His Bind family of products offers a wide selection of supports designed for rapid onestep purification of proteins containing the His Tag sequence by immobilized metal affinity chromatography (IMAC). The various His Bind supports listed in the table cover many applications for His Tag fusion protein purification. Choices include bulk easy-tohandle agaroses for batch and gravity flow columns, small scale cellulose-based columns and cartridges for convenient handling of multiple samples, and high flow rate Superflow and Fractogel resins suitable for production scale purification. Several supports are provided pre-charged with Ni 2+. s for both NTA and IDA Ni 2+ chelation chemistries are available. Chelator Nitrilotriacetic Acid (NTA) Iminodiacetic Acid (IDA) Ni-NTA His Bind Resin Ni-NTA His Bind Superflow His Bind Resin His Bind Magnetic Agarose Beads His Bind Column Form/Resin Composition Ni-charged cross-linked beaded agarose; 50% slurry in 30% ethanol Ni-charged 6% cross-linked beaded agarose; 50% slurry in 30% ethanol Uncharged cross-linked beaded agarose; 50% slurry in 20% ethanol Ni-charged polystyrene coated ferric oxide; 50% suspension Ni-charged cross-linked beaded agarose; 1.25 ml packed column Capacity 5 10 mg/ml 5 10 mg/ml 8 mg/ml 5 mg/ml 10 mg/run His Bind Quick Resin Ni-charged beaded cellulose, dry resin 2.5 mg/ml Ni-NTA His Bind Resin* ml ml ml His Bind Quick 300 Cartridge His Bind Quick 900 Cartridge cartridge, dry resin cartridge, dry resin 0.5 mg/run 2 mg/run Ni-NTA His Bind Superflow * ml ml ml His Bind Resin ml ml ml His Bind Magnetic Agarose Beads* ml ml His Bind Quick Column His Bind Fractogel (S) His Bind Fractogel (M) manufactured by QIAGEN column, dry resin Uncharged tentacle methacrylate polymer; suspension in 20% ethanol and 150 mm NaCl Uncharged tentacle methacrylate polymer; suspension in 20% ethanol and 150 mm NaCl 5 mg/run > 10 mg/ml > 10 mg/ml * resin is pre-charged with Ni Protein Purification

2 NTA AND IDA NI 2+ CHELATION CHEMISTRIES With the His Tag /His Bind technology, purification is based on the affinity between neighboring histidines of the His Tag sequence and an immobilized metal ion (usually Ni 2+ ). The metal is held by chelation with reactive groups covalently attached to a solid support. The most commonly used chelators include nitrilotriacetic acid (NTA) and iminodiacetic acid (IDA), which have four and three sites available for interaction with metal ions, respectively. The two chemistries confer different properties to the affinity support and conditions used for binding, washing and elution of target proteins for both native and denaturing conditions. In practice, the additional chelation site available with NTA minimizes leaching of the metal during the purification and is compatible with up to 20 mm 2-mercaptoethanol for reduction of disulfide bonds. The higher metal leaching rates of IDA-based resins in the presence of other chelating or reducing components can produce poor purification results. However, IDA supports can be recycled many times with no loss in performance. For both types of support the conditions can be modified to optimize the purification of individual His Tag fusion proteins expressed in specific systems. In most applications, the imidazole concentrations of the wash and elution buffers under native conditions can be adjusted to minimize co-purification of non-specifically bound proteins. His Bind Columns* pkg/ pkg/ His Bind Quick Resin* RT bulk inquire Particle Size (µm) Max. Pressure 140 psi Features Minimal Ni 2+ leaching. Compatible with 20 mm 2-ME and Ni-NTA Buffer Kit Minimal Ni 2+ leaching. Compatible with 20 mm 2-ME and Ni-NTA Buffer Kit. High flow rates and pressures Reusable many times. Compatible with His Bind. Buffer Kit Applications Small to medium scale. Gravity flow column. Recommended for eukaryotic extracts. FPLC or gravity flow column. Recommended for eukaryotic extracts. Small to medium scale. Gravity flow column or batch mode His Bind Quick 300 Cartridges* RT pkg/ pkg/ His Bind Quick 900 Cartridges* RT pkg/ pkg/ His Bind Quick Columns* RT pkg/ pkg/ Average 3 µm 3 µm magnetic agarose beads Rapid small scale purification. Magnetic separation. HT compatible His Bind Fractogel (S) ml Compatible with His Bind Buffer Kit Convenient purification. Gravity flow column His Bind Fractogel (M) ml Compatible with His Bind Quick Buffer Kit Small to large scale purification * resin is pre-charged with Ni 2+ Luer fitting on both ends. Compatible with His Bind Quick Buffer Kit Syringe-driven processing. Rapid purification Luer fitting on both ends Compatible with His Bind Quick Buffer Kit Syringe-driven processing. Rapid purification Compatible with His Bind Quick Buffer Kit Vacuum Manifold processing. Rapid purification of multiple samples psi High flow rates and pressures Small to production scale. FPLC or gravity flow column. High resolution separations psi High flow rates and pressures Small to production scale. FPLC or gravity flow column RT = room temperature (+20 C) = refrigeration (+4 C) = freezer ( 20 C) = multiple storage temperatures = deep-freeze ( 70 C) = shipped with blue ice = shipped with dry ice = shipped as hazardous 23

3 HIS BIND AND HIS BIND QUICK BUFFER KITS These kits provide a convenient and rapid one-step purification of proteins by metal chelation chromatography. The His Bind Buffer Kit is a set of pre-tested buffers designed for use with IDA-based His Bind Resin. Solutions are included for Ni 2+ charging, binding, washing and elution of up to ten 2.5 ml columns. The His Bind Quick Buffer Kit is designed for use with the pre-charged His Bind Quick resins and contains all the components of the His Bind Buffer Kit except the 8X Charge Buffer. NI-NTA BUFFER KIT The Ni-NTA Buffer Kit provides a convenient set of buffers optimized for purification of His Tag fusion proteins on Ni-NTA His Bind Resin under native conditions. These phosphate-buffered solutions differ from the Tris-based solutions used in the His Bind Buffer Kit. Carefully prepared 4X concentrates are included for binding, washing and elution according to recommended protocols. His Bind Buffer Kit kit His Bind Quick Buffer Kit kit Ni-NTA Buffer Kit kit His Bind kit BugBuster Ni-NTA His Bind kit BUGBUSTER NI-NTA HIS BIND AND HIS BIND PURIFICATION KITS BugBuster His Bind kit BugBuster Protein Extraction Reagent is a ready-to-use solution that efficiently extracts soluble protein from E. coli without the need for mechanical disruption. The BugBuster Ni-NTA His Bind and His Bind s combine BugBuster reagent with the respective resins for convenient preparation of soluble cell extracts and affinity purification of His Tag fusion proteins. Both kits include Benzonase Nuclease for viscosity reduction and removal of nucleic acids from protein preparations. The BugBuster His Bind also includes buffers for chromatography M His Bind purification of a poorly expressed protein A target protein comprising less than 5% of the total protein prepared from a recombinant expressed in pet- 32b(+) was purified using the His Bind Resin and His Bind Buffer Kit. The high specificity of the His Bind Resin is demonstrated by analysis of the indicated fractions by SDS-PAGE and Coomassie blue staining. Lanes: 1, total protein; 2, column flow-through; 3, wash; 4, elution; M, Protein Markers. 24 Protein Purification

4 GST TAG, S TAG AND T7 TAG PURIFICATION RESINS AND KITS Capacity Comments M GST Bind Resin 5 8 mg/ml GST Bind Resin utilizes an 11-atom spacer arm to covalently attach reduced glutathione via a sulfide linkage. The resin can be re-used several times without loss of binding capacity. GST Bind Magnetic Agarose Beads 5 8 mg/run Glutathione attached to magnetic agarose beads for rapid small scale purification. HT compatible. GST Bind Buffer Kit Kit provides reagents needed for optimized binding, washing and elution of target proteins from GST Bind Resin. Sufficient components are provided to run a minimum of ten 2.5 ml columns. BugBuster GST Bind S-protein Agarose 0.5 mg/ml Kit combines the BugBuster Protein Extraction Reagent and Benzonase Nuclease with both GST Bind Resin and GST Bind Buffers in one convenient kit. BugBuster Protein Extraction Reagent is formulated for the gentle disruption of the cell wall of E. coli, resulting in the liberation of soluble protein. S-protein Agarose specifically retains S Tag fusion proteins GST Bind purification A crude extract containing excess unfused GST was applied to a 2 ml GST Bind Resin column. Total protein yield after purification was 8 mg/ml resin. Lanes: M, Perfect Protein Markers; 1, Crude extract; 2, Flowthrough; 3, Eluate S Tag Thrombin S Tag rek Purification Kit T7 Tag Antibody Agarose T7 Tag Affinity 0.3 mg/ml M Kit is designed for purification of S Tag fusion proteins that contain a thrombin cleavage site (LeuValProArg GlySer) between the S Tag sequence and the cloning region. Includes Biotinylated Thrombin that can be removed using Streptavidin Agarose. Streptavidin Agarose is provided with the kit. Kit is designed for purification of S Tag fusion proteins that contain an enterokinase cleavage site (AspAspAspAspLys ) between the S Tag sequence and the cloning region. The kit enables target protein release by incubation of the agarose beads with Recombinant Enterokinase (rek), leaving the S Tag peptide bound to the resin. Residual rek can then be removed efficiently using EKapture Agarose beads. EKapture Agarose beads are provided with the kit. T7 Tag Monoclonal Antibody covalently coupled to crosslinked agarose beads. Kit is based on binding T7 Tag fusion proteins to T7 Tag Antibody Agarose. Capacity will vary somewhat between different target proteins, but the beads are standardized to bind a minimum of 300 µg T7 Tag β-galactosidase per milliliter of settled resin. The beads can be used in either batch or column methods and can be recycled a minimum of five times without loss of binding activity. M GST Bind Resin ml ml GST Bind Magnetic Agarose Beads ml ml GST Bind Buffer Kit kit BugBuster GST Bind kit S-protein Agarose ml x 2 ml S Tag Thrombin kit S Tag rek kit S Tag affinity purification S Tag β-galactosidase expressed from a pet construct was purified from a crude soluble fraction using S-protein Agarose under native conditions. Elution was performed by digestion with Biotinylated Thrombin. Lanes: M, Perfect Protein Markers; 1, Crude extract; 2, Flow through; 3, Wash 1; 4, Wash 2; 5, Eluate + B-Thrombin; 6, Eluate after B-Thrombin removal. T7 Tag affinity purification T7 Tag β-galactosidase expressed from a pet construct was purified from a crude soluble fraction using T7 Tag Agarose under native conditions. The target protein was eluted and samples analyzed by SDS-polyacrylamide gel electrophoresis (4 20% gradient gel) and Coomassie blue staining. Lanes: M, Perfect Protein Markers; 1, Crude extract; 2, Flow through; 3, Eluate. RT = room temperature (+20 C) = refrigeration (+4 C) = freezer ( 20 C) = multiple storage temperatures = deep-freeze ( 70 C) = shipped with blue ice = shipped with dry ice = shipped as hazardous T7 Tag Antibody Agarose x 1 ml T7 Tag Affinity kit

5 Enzyme Thrombin Thrombin, Restriction Grade Comments Restriction Grade Thrombin specifically cleaves target proteins containing the recognition sequence LeuValProArg GlySer. Thrombin is supplied with 10X Thrombin Cleavage Buffer and a Cleavage Control Protein. Thrombin, Restriction Grade U Biotinylated Thrombin Biotinylated Thrombin is Restriction Grade Thrombin covalently attached to biotin. It can be easily removed from cleavage reactions using immobilized streptavidin. Has 99% binding to Streptavidin Agarose. Biotinylated Thrombin U Thrombin Cleavage kit Factor Xa, Restriction Grade U Thrombin Cleavage The Thrombin Cleavage is designed for cleavage of fusion proteins followed by removal of thrombin protease. Kit is suitable for use with any fusion protein that contains a thrombin recognition sequence. Kit includes Biotinylated Thrombin, Streptavidin Agarose, Spin Filters and a Cleavage Control Protein to monitor efficiency of thrombin cleavage. Factor Xa Cleavage kit Recombinant Enterokinase U Factor Xa Factor Xa, Restriction Grade Restriction Grade Factor Xa is a highly purified preparation isolated from bovine plasma and activated with Russell s viper venom. Factor Xa cleaves at the C-terminal side of the recognition sequence (IleGluGlyArg ) and can be used for removing all vector-encoded sequences from appropriately designed constructs. Enterokinase Cleavage kit Factor Xa Cleavage The Factor Xa Cleavage is designed for highly specific cleavage of fusion proteins and convenient removal of Factor Xa. Factor Xa is efficiently removed by Xarrest Agarose. No buffer changes are necessary. The kit includes a Cleavage Control Protein for control digests. Enterokinase Recombinant Enterokinase Highly purified Recombinant Enterokinase (rek) recognizes the identical cleavage site as the native enzyme (AspAspAspAspLys ) and has similar enzymatic activity. rek exhibits superior rates of cleavage of fusion proteins containing the recognition sequence when compared to the native enzyme. Enterokinase Cleavage The Enterokinase Cleavage is designed for highly specific cleavage of fusion proteins followed by rapid, affinity-based removal of enterokinase. rek is removed by capture on EKapture Agarose. No buffer changes are necessary. Cleavage Control Protein is included in the kit. 26 Protein Purification