Bio-rad Nuvia™ IMAC Resin Manual de usuario

Bio-Rad® Nuvia™ IMACResinInstruction ManualCatalog numbers780-0800780-0801780-0802Please read these instructions prior to using Bio-Rad Nuvia IMAC res

6 Nuvia IMAC Ni-Charged ResinTable 2. Chemical Compatibilities for Nuvia IMAC Resins, Continued.Reagent GroupReagent Comments StabilityOther additi

Nuvia IMAC Ni-Charged Resin 7 Section 3General IMAC ProceduresProtein BindingProtein adsorption to immobilized ions is performed around neutral to s

8 Nuvia IMAC Ni-Charged ResinWashesStringency of 6x histidine-tag binding can be effectively increased by 1) including low concentrations of imidaz

Nuvia IMAC Ni-Charged Resin 9 If lower pH is used to elute bound proteins, tubes filled with a strong neutralizing buffer such as 1 M Tris-HCl, pH 8.

10 Nuvia IMAC Ni-Charged ResinFinally, the use of a liquid chromatography system ensures that the adjustment of denaturants, detergents, salts, and

Nuvia IMAC Ni-Charged Resin 11 Section 4Column Packing — Medium-Pressure ColumnsSlurry Packing an IMAC Column Slurry packing is preferred for small

12 Nuvia IMAC Ni-Charged ResinResin Preparation Nuvia IMAC Ni-charged resins come in a 20% ethanol solution, supplied for resin storage. The column

Nuvia IMAC Ni-Charged Resin 13 7. Adjust the adaptor to sit directly on top of the resin bed.Open the column outlet and pump distilled buffer throu

14 Nuvia IMAC Ni-Charged ResinSection 5Column Packing — Sample Preparation–Sized ColumnsUse this method for packing Nuvia™ IMAC resin into sample p

Nuvia IMAC Ni-Charged Resin 15 Method 1. Thoroughly suspend Nuvia IMAC resin.2. Place the column into an appropriate collection vessel; for examp

16 Nuvia IMAC Ni-Charged ResinSection 6Immobilizing Metal IonsEfficacy of protein binding by IMAC depends on two factors: the number of available hi

Nuvia IMAC Ni-Charged Resin 17 Section 7Medium-Pressure Column Purification of Histidine-Tagged ProteinsFor this guideline, the sample is applied to

18 Nuvia IMAC Ni-Charged Resin2. Equipment• IMAC column (as prepared in Section 4)3. Biological Sample• Clarified lysateThe binding capacity of

Nuvia IMAC Ni-Charged Resin 19 5. Collect fractions from wash steps.Pool recovered unbound proteins with fractions collected in step 3.6. Elute bo

20 Nuvia IMAC Ni-Charged ResinMaterials for On-Column Renaturation 1. ReagentsRefolding buffer• 20 mM sodium phosphate (NaH2PO4) (pH 8.0)• 20 mM

Nuvia IMAC Ni-Charged Resin 21 Section 8Medium-Pressure Column Purification — Using an Imidazole Gradient to Determine Optimal Purification of Histidi

22 Nuvia IMAC Ni-Charged Resin3. Biological Sample• Clarified lysateNote: Keep the sample as small as possible during optimization of binding and

Nuvia IMAC Ni-Charged Resin 23 8. Equilibrate the column with 10 column volumes of binding buffer (0%B). Stop collecting fractions.9. Identify the

24 Nuvia IMAC Ni-Charged ResinPart 2: Using an Optimized Imidazole Concentration for Purification1. Prepare 500 ml binding buffer and 500 ml elutio

Nuvia IMAC Ni-Charged Resin 25 Section 9Sample Preparation–Sized Spin-Column Purification of Histidine-Tagged ProteinsMaterials 1. ReagentsBinding/

Table of ContentsSection 1 Introduction ... 1Section 2 Product Description ...

26 Nuvia IMAC Ni-Charged Resin3. Biological Sample• Clarified lysateThe binding capacity of Nuvia™ IMAC resin is ≥40 mg histidine-tagged protein p

Nuvia IMAC Ni-Charged Resin 27 Part 2: Washing the Resin1. Insert micro spin column into new collection vessel.2. Wash the resin with at least 5 c

28 Nuvia IMAC Ni-Charged ResinSection 10Regenerating, Cleaning, Sanitizing, and StorageNuvia™ IMAC columns are well-suited for reuse. The polymeric

Nuvia IMAC Ni-Charged Resin 29 1. Strip metal ions.Wash with 10 column volumes of 50 mM sodium phosphate, 0.3 M NaCl, and 0.05–0.5 M EDTA, pH 7.5.2

30 Nuvia IMAC Ni-Charged ResinSanitizationSanitization inactivates microorganisms and prevents buildup of endotoxins. For optimal results, the colu

Nuvia IMAC Ni-Charged Resin 31 Section 11Troubleshooting Guide Problem Possible Cause SolutionSample is too viscous High concentration of host nucle

32 Nuvia IMAC Ni-Charged ResinProblem Possible Cause SolutionNo protein is eluted (continued from previous page)Target protein is found in the flowt

Nuvia IMAC Ni-Charged Resin 33 Problem Possible Cause SolutionHistidine-tagged protein is not pure (continued from previous page)Association between

34 Nuvia IMAC Ni-Charged ResinSection 12Ordering InformationNuvia IMAC™ Ni-Charged ResinCatalog # Description780-0800 Nuvia IMAC Ni-Charged Resin,

Section 8 Medium-Pressure Column Purification — Using an Imidazole Gradient to Determine Optimal Purification of Histidine-Tagged Proteins ...

Life ScienceGroupSig 121310044307 Rev A US/EGBio-Rad Laboratories, Inc.Web site www.bio-rad.com USA 800 424 6723 Australia 61 2 9914 2800 Austr

Nuvia IMAC Ni-Charged Resin 1 Section 1IntroductionImmobilized metal affinity chromatography (IMAC) is a powerful purification technique that relies o

2 Nuvia IMAC Ni-Charged ResinSection 2Product InformationNuvia™ IMAC Resins and UNOsphere™ TechnologyNuvia IMAC resin, a unique affinity support, is

Nuvia IMAC Ni-Charged Resin 3 Under optimized conditions, the binding capacity for 6x histidine-tagged proteins is >40 mg/ml resin (see Table 1).

4 Nuvia IMAC Ni-Charged ResinResin CharacteristicsThe characteristics of Nuvia IMAC resin are detailed in Table 1.Table 1. Characteristics of Bio-R

Nuvia IMAC Ni-Charged Resin 5 Chemical CompatibilitiesThe chemical characteristics of Nuvia IMAC resin are detailed in Table 2.Table 2. Chemical Com