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Clontech Laboratories His60 Ni Superflow Cartridges User Manual

Purification of expressed his-tagged proteins from bacterial, yeast, mammalian, and baculovirus-infected cells.
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Clontech Laboratories, Inc.
His60 Ni Superflow
Cartridges User
Manual
PT5143-1
Cat. No(s). 635674, 635675, 635678, 635679, 635680
(031716)
Clontech Laboratories, Inc.
A Takara Bio Company
1290 Terra Bella Avenue, Mountain View, CA 94043, USA
U.S. Technical Support:
tech@clontech.com
United States/Canada
Asia Pacific
800.662.2566
+1.650.919.7300
Europe
Japan
+33.(0)1.3904.6880
+81.(0)77.543.6116

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Summary of Contents for Clontech Laboratories His60 Ni Superflow Cartridges

  • Page 1 Clontech Laboratories, Inc. His60 Ni Superflow Cartridges User Manual PT5143-1 Cat. No(s). 635674, 635675, 635678, 635679, 635680 (031716) Clontech Laboratories, Inc. A Takara Bio Company 1290 Terra Bella Avenue, Mountain View, CA 94043, USA U.S. Technical Support: tech@clontech.com United States/Canada...

  • Page 2: Table Of Contents

    A. PROTOCOL: Extracting Proteins from Cells ......................10 PROTOCOL: Automated Purification on a Liquid Chromatography System ............11 PROTOCOL: Manual Purification Using a Syringe ....................12 D. PROTOCOL: Complete Regeneration of His60 Ni Superflow Cartridges ............... 14 Troubleshooting Guide ............................. 15 Table of Tables Table 1.

  • Page 3: Introduction

    His60 Ni Superflow Cartridges User Manual Introduction Clontech’s His60 Ni Superflow Cartridges are prefilled with 1 ml or 5 ml of His60 Ni Superflow resin and are ready to use for purification of his-tagged proteins using a syringe, peristaltic pump, or liquid chromatography systems such as ÄKTA or other FPLC systems.

  • Page 4: His60 Ni Superflow Resin

    (also available separately as the His60 Ni Buffer Set)—see Section II. The His60 Ni Cartridge Purification Kit (5 x 5 ml) provides 5 prepacked His60 Ni Superflow Cartridges (each containing 5 ml of resin), as well as all the buffers needed for protein extraction and purification (also available separately as the His60 Ni Buffer Set)—see Section II.

  • Page 5: Table 2. Reagent Compatibility With His60 Ni Superflow Resin (Based On Literature References)

    His60 Ni Superflow Cartridges User Manual Table 2. Reagent Compatibility with His60 Ni Superflow Resin (Based on Literature References) Reagent Notes Acceptable Concentrations Amino Acids Arginine, Glycine, Not recommended Glutamine Histidine Binds to His60 Ni and competes with histidine residues in the Can be used at low concentrations (20 his-tag.

  • Page 6: List Of Components

    635656 & 635671 (see Section IV). His60 Ni Superflow Cartridge Purification Kit (5 x 1 ml) (Cat. No. 635674)  1 His60 Ni Superflow Cartridges (5 x 1 ml) (Cat. No. 635675)  1 His60 Ni Buffer Set (Cat. No. 635665) His60 Ni Superflow Cartridge (1 x 5 ml) (Cat.

  • Page 7: Additional Materials Required

    Elution Buffers, the His60 Ni Buffer Set (Cat. No.635665), which is sufficient for approximately 20 purifications on 1 ml His60 Ni Superflow Cartridges (5 x 1 ml) (Cat. No.635675), or 5 purifications on 5 ml His60 Ni Superflow Cartridges (5 x 5 ml) (Cat. No. 635679).

  • Page 8: Buffers - Denaturing Conditions - Guanidine-Hcl Or Urea

    His60 Ni Superflow Cartridges User Manual C. Buffers – Denaturing Conditions – Guanidine-HCl or Urea Denaturants, such as 5 M guanidine-HCl or 8 M urea, enhance protein solubility. Because overexpressed proteins in prokaryotic systems are sometimes insoluble, you may need to purify proteins under denaturing conditions.

  • Page 9: Related Products: Extraction Buffers, Protease Inhibitors, And His-Tag Antibodies

    His60 Ni Superflow Cartridges User Manual Related Products: Extraction Buffers, Protease Inhibitors, and His-Tag Antibodies (Cat. No. 635623) xTractor Buffer Kit Applications: extraction of insoluble protein from inclusion bodies, efficient extraction of high molecular weight proteins, and complete disruption of bacterial cell walls and membranes ...

  • Page 10: Sample Preparation & Purification

    His60 Ni Superflow Cartridges User Manual Sample Preparation & Purification PLEASE READ THE ENTIRE PROTOCOL BEFORE STARTING. Use this procedure to (a) prepare your his-tagged protein sample for (b) automated or (c) manual purification using His60 Ni Superflow Cartridges. PROTOCOL: Extracting Proteins from Cells This procedure has been optimized for extraction of proteins from fresh or frozen cell pellets using xTractor Buffer.

  • Page 11: Protocol: Automated Purification On A Liquid Chromatography System

    His60 Ni Superflow Cartridges User Manual Lysate clarification Centrifuge the crude lysate at at 4°C. Carefully transfer the 10,000–12,000 x g 20 min supernatant to a clean tube without disturbing the pellet. This is your starting sample. NOTE: If the supernatant is not clear, centrifuge a second time or filter through a 0.45 µm membrane (e.g., cellulose acetate) to avoid clogging the IMAC column with insoluble...

  • Page 12: Protocol: Manual Purification Using A Syringe

    His60 Ni Superflow Cartridges User Manual Wash Step Wash the cartridge using a flow rate of for 1 ml cartridges or for 5 ml 1 ml/min 5 ml/min cartridges with followed by 8 column volumes Equilibration Buffer 7 column volumes (i.e., Equilibration Buffer containing 40 mM imidazole).
  • Page 13 His60 Ni Superflow Cartridges User Manual Cartridge Setup a. Attach the syringe to a Luer Lock Adapter (not supplied; see Section III.F). b. Remove the top plug from the cartridge, add a few drops of buffer from the syringe to the top inlet of the cartridge, and attach the syringe to the top of the cartridge via the Luer Lock adapter.

  • Page 14: Protocol: Complete Regeneration Of His60 Ni Superflow Cartridges

    20% ethanol 4°C Attach supplied bottom cap, followed by the top plug. D. PROTOCOL: Complete Regeneration of His60 Ni Superflow Cartridges If you plan to purify multiple proteins using the same column, you must use the following resin regeneration protocol before you purify a new protein.

  • Page 15: Troubleshooting Guide

    Clontech®, the Clontech logo, ProteoGuard, TALON®, and xTractor are trademarks of Clontech Laboratories, Inc. All other trademarks are the property of their respective owners. Certain trademarks may not be registered in all jurisdictions. Clontech Laboratories, Inc. is a Takara Bio Company. ©2016 Clontech Laboratories, Inc.

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