重组蛋白纯化说明书解析.docx

1、重组蛋白纯化说明书解析 Instruction ManualProBond TM Purification SystemFor purification of polyhistidine-containing recombinant proteinsCatalog nos. K850-01, K851-01, K852-01, K853-01, K854-01, R801-01, R801-15Version K2 September 200425-0006iiTable of ContentsKit Contents and Storage.iv Accessory Products.vi

2、Introduction.1 Overview.1 Methods.2 Preparing Cell Lysates.2 Purification ProcedureNative Conditions.7 Purification ProcedureDenaturing Conditions.11 Purification ProcedureHybrid Conditions.13 Troubleshooting.15 Appendix.17 Additional Protocols.17 Recipes.18 Frequently Asked Questions.21 References.

3、22 Technical Service.23iiiKit Contents and StorageTypes of Products This manual is supplied with the following products:Product Catalog No. ProBond Purification System K850-01ProBond Purification System with Antibodywith Anti-Xpress Antibody K851-01with Anti-myc-HRP Antibody K852-01with Anti-His(C-t

4、erm-HRP Antibody K853-01with Anti-V5-HRP AntibodyK854-01ProBond Nickel-Chelating Resin (50 ml R801-01ProBond Nickel Chelating Resin (150 ml R801-15ProBond Purification System Components The ProBond Purification System includes enough resin, reagents, and columns for six purifications. The components

5、 are listed below. See next page for resin specifications.ProBond Resin 50% slurry in 20% ethanol 12 ml 5X NativePurification Buffer250 mM NaH2PO 4, pH 8.02.5 M NaCl1 125 ml bottleGuanidinium LysisBuffer6 M Guanidine HCl20 mM sodium phosphate, pH 7.8500 mM NaCl1 60 ml bottleDenaturingBinding Buffer8

6、 M Urea20 mM sodium phosphate, pH 7.8500 mM NaCl2 125 ml bottlesDenaturing WashBuffer8 M Urea20 mM sodium phosphate, pH 6.0500 mM NaCl2 125 ml bottlesDenaturing ElutionBuffer8 M Urea20 mM NaH2PO 4, pH 4.0500 mM NaCl1 60 ml bottleImidazole 3 M Imidazole,20 mM sodium phosphate, pH 6.0500 mM NaCl1 8 ml

7、 bottlePurificationColumns10 ml columns 6Continued on next pageivKit Contents and Storage, ContinuedProBond Purification System with Antibody The ProBond Purification System with Antibody includes resin, reagents, and columns as described for the ProBond Purification System (previous page and 50 l o

8、f 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 protocols for using the antibody, refer to the antibody manual supplied with the syst

9、em.Storage Store ProBond resin at +4C. Store buffer and columns at room temperature. Store the antibody at 4C. Avoid repeated freezing and thawing of theantibody as it may result in loss of activity.The product is guaranteed for 6 months when stored properly. All native purification buffers are prep

10、ared 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 ColumnSpecificationsProBond resin is precharged with Ni2+ ions and appears blue in color.

11、 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: 45165 microns Pore size of purification columns: 3035 microns Recommended flow rate: 0.5 ml/

12、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 214ProductQualificationThe ProBond Purification System is qualified by purifying 2 mg of myoglobin protein on a column and performing a Bradf

13、ord assay. Protein recovery must be 75% or higher.vAccessory ProductsAdditionalProductsThe following products are also available for order from Invitrogen:Product QuantityCatalog No.ProBond Nickel-Chelating Resin 50 ml150 mlR801-01 R801-15Polypropylene columns(empty50 R640-50Ni-NTA Agarose 10 ml 25

14、ml R901-01 R901-15Ni-NTA Purification System 6 purifications K950-01 Ni-NTA Purification Systemwith Antibodywith Anti-Xpress Antibody with Anti-myc-HRP Antibody with Anti-His(C-term-HRP Antibodywith Anti-V5-HRP Antibody 1 kit1 kit1 kit1 kitK951-01 K952-01 K953-01 K954-01Anti-myc Antibody 50 l R950-2

15、5 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-gelStaining Kit1 kit LC6033Pre-Cast Gels and Pre-made Buffers A large variety of pre-cast gels for SDS-PAGE and pre-made buffers for

16、 your convenience are available from Invitrogen. For details, visit our web site at or contact Technical Service (page 23.viIntroductionOverviewIntroduction The ProBond Purification System is designed for purification of 6xHis-tagged recombinant proteins expressed in bacteria, insect, and mammalian

17、cells. The system is designed around the high affinity and selectivity of ProBond Nickel-Chelating Resin for recombinant fusion proteins containing six tandem histidine residues.The ProBond Purification System is a complete system that includespurification buffers and resin for purifying proteins un

18、der native, denaturing, or hybrid conditions. The resulting proteins are ready for use in many target applications.This manual is designed to provide generic protocols that can be adapted for your particular proteins. The optimal purification parameters will vary with each protein being purified.Pro

19、Bond Nickel-Chelating Resin ProBond Nickel-Chelating Resin is used for purification of recombinant proteins expressed in bacteria, insect, and mammalian cells from any 6xHis-tagged vector. ProBond Nickel-Chelating Resin exhibits high affinity and selectivity for 6xHis-tagged recombinant fusion prote

20、ins.Proteins can be purified under native, denaturing, or hybrid conditions using the ProBond Nickel-Chelating Resin. Proteins bound to the resin are eluted with low pH buffer or by competition with imidazole or histidine. The resulting proteins are ready for use in target applications.Binding Chara

21、cteristics ProBond Nickel-Chelating Resin uses the chelating ligand iminodiacetic acid (IDA in a highly cross-linked agarose matrix. IDA binds Ni2+ ions by three coordination sites. The protocols provided in this manual are generic, and may not result in 100% pure protein. These protocols should be

22、optimized based on the binding characteristics of your particular proteins.Native VersusDenaturingConditionsThe decision to purify your 6xHis-tagged fusion proteins under native or denaturing conditions depends on the solubility of the protein and the need to retain biological activity for downstrea

23、m applications. Use native conditions if your protein is soluble (in the supernatant after lysis and you want to preserve protein activity. Use denaturing conditions if the protein is insoluble (in the pellet after lysis or if your downstream application does not depend on protein activity. Use hybr

24、id protocol if your protein is insoluble but you want to preserve protein activity. Using this protocol, you prepare the lysate and columns under denaturing conditions and then use native buffers during the wash and elution steps to refold the protein. Note that this protocol may not restore activit

25、y for all proteins. See page 14.1MethodsPreparing Cell LysatesIntroduction Instructions for preparing lysates from bacteria, insect, and mammalian cells using native or denaturing conditions are described below.Materials Needed You will need the following items: Native Binding Buffer (recipe is on page 8 for preparing lysat