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1、Instruction Manual ProBond TM Purification System For purification of polyhistidine-containing recombinant proteins Catalog nos. K850-01, K851-01, K852-01, K853-01, K854-01, R801-01, R801-15 Version K 2 September2004 25-0006 ii iii Table of Contents Kit Contents and Storageiv Accessory Products.vi I
2、ntroduction.1 Overview 1 Methods.2 Preparing Cell Lysates2 Purification ProcedureNative Conditions7 Purification ProcedureDenaturing Conditions .11 Purification ProcedureHybrid Conditions.13 Troubleshooting.15 Appendix17 Additional Protocols.17 Recipes 18 Frequently Asked Questions21 References.22 T
3、echnical Service .23 iv Kit Contents and Storage Types of Products This manual is supplied with the following products: Product Catalog No. ProBond Purification System K850-01 ProBond Purification System with Antibody with Anti-Xpress Antibody K851-01 with Anti-myc-HRP Antibody K852-01 with Anti-His
4、(C-term)-HRP Antibody K853-01 with Anti-V5-HRP Antibody K854-01 ProBond Nickel-Chelating Resin (50 ml) R801-01 ProBond Nickel Chelating Resin (150 ml) R801-15 ProBond Purification System Components The ProBond Purification System includes enough resin, reagents, and columns for six purifications. Th
5、e components are listed below. See next page for resin specifications. Component Composition Quantity ProBond Resin 50% slurry in 20% ethanol 12 ml 5X Native Purification Buffer 250 mM NaH2PO4, pH 8.0 2.5 M NaCl 1 125 ml bottle Guanidinium Lysis Buffer 6 M Guanidine HCl 20 mM sodium phosphate, pH 7.
6、8 500 mM NaCl 1 60 ml bottle Denaturing Binding Buffer 8 M Urea 20 mM sodium phosphate, pH 7.8 500 mM NaCl 2 125 ml bottles Denaturing Wash Buffer 8 M Urea 20 mM sodium phosphate, pH 6.0 500 mM NaCl 2 125 ml bottles Denaturing Elution Buffer 8 M Urea 20 mM NaH2PO4, pH 4.0 500 mM NaCl 1 60 ml bottle
7、Imidazole 3 M Imidazole, 20 mM sodium phosphate, pH 6.0 500 mM NaCl 1 8 ml bottle Purification Columns 10 ml columns 6 Continued on next page v Kit Contents and Storage, Continued ProBond Purification System with Antibody The ProBond Purification System with Antibody includes resin, reagents, and co
8、lumns as described for the ProBond Purification System (previous page) and 50 l of the appropriate purified mouse monoclonal antibody. Sufficient reagents are included to perform six purifications and 25 Western blots with the antibody. For more details on the antibody specificity, subclass, and pro
9、tocols for using the antibody, refer to the antibody manual supplied with the system. Storage Store ProBond resin at +4C. Store buffer and columns at room temperature. Store the antibody at 4C. Avoid repeated freezing and thawing of the antibody as it may result in loss of activity. The product is g
10、uaranteed for 6 months when stored properly. All native purification buffers are prepared from the 5X Native Purification Buffer and the 3 M Imidazole, as described on page 7. The Denaturing Wash Buffer pH 5.3 is prepared from the Denaturing Wash Buffer (pH 6.0), as described on page 11. Resin and C
11、olumn Specifications ProBond resin is precharged with Ni2+ ions and appears blue in color. It is provided as a 50% slurry in 20% ethanol. ProBond resin and purification columns have the following specifications: Binding capacity of ProBond resin: 15 mg of protein per ml of resin Average bead size: 4
12、5165 microns Pore size of purification columns: 3035 microns Recommended flow rate: 0.5 ml/min Maximum flow rate: 2 ml/min Maximum linear flow rate: 700 cm/h Column material: Polypropylene pH stability (long term): pH 313 pH stability (short term): pH 214 Product Qualification The ProBond Purificati
13、on System is qualified by purifying 2 mg of myoglobin protein on a column and performing a Bradford assay. Protein recovery must be 75% or higher. vi Accessory Products Additional Products The following products are also available for order from Invitrogen: Product Quantity Catalog No. ProBond Nicke
14、l-Chelating Resin 50 ml 150 ml R801-01 R801-15 Polypropylene columns (empty) 50 R640-50 Ni-NTA Agarose 10 ml 25 ml R901-01 R901-15 Ni-NTA Purification System 6 purifications K950-01 Ni-NTA Purification System with Antibody with Anti-Xpress Antibody with Anti-myc-HRP Antibody with Anti-His(C-term)-HR
15、P Antibody with Anti-V5-HRP Antibody 1 kit 1 kit 1 kit 1 kit K951-01 K952-01 K953-01 K954-01 Anti-myc Antibody 50 l R950-25 Anti-V5 Antibody 50 l R960-25 Anti-Xpress Antibody 50 l R910-25 Anti-His(C-term) Antibody 50 l R930-25 InVision His-tag In-gel Stain 500 ml LC6030 InVision His-tag In-gel Stain
16、ing Kit 1 kit LC6033 Pre-Cast Gels and Pre-made Buffers A large variety of pre-cast gels for SDS-PAGE and pre-made buffers for your convenience are available from Invitrogen. For details, visit our web site at or contact Technical Service (page 23). 1 Introduction Overview Introduction The ProBond Purification System is designed for purification of 6xHis-tagged recombinant proteins expressed in bacteria, insect, and mamm
