Thermo Fisher Scientific Pierce User Manual

USER GUIDE

Pierce™ High-Capacity Ni-IMAC Magnetic Beads, EDTA
Compatible

Catalog Numbers A50588, A50589, A50590, and A50591

Pub. No. MAN0024861 Rev. A.0

WARNING! Read the Safety Data Sheets (SDSs) and follow the handling instructions. Wear appropriate protective eyewear,

clothing, and gloves. Safety Data Sheets (SDSs) are available from thermofisher.com/support.

Product description

The Pierce™ High-Capacity Ni-IMAC Magnetic Beads, EDTA Compatible are ferrimagnetic agarose beads coupled to a novel, proprietary
ligand loaded with nickel ions. The beads enable ecient purification of recombinant polyhistidine-tagged proteins from a soluble protein
extract or mammalian cell culture supernatant and are compatible with native or denaturing conditions. They can be used in manual
applications with a magnetic stand or automated applications with an instrument such as the Thermo Fisher™ KingFisher™ Flex System.
They have high binding anity for His-tagged proteins and low-metal, ion-leaching characteristics, even in the presence of chemical
additives including chelators (EDTA), strong reducing agents (DTT), or components of cell culture supernatants that typically strip o Ni
ions and reduce the functionality of most IMAC magnetic beads.

Table 1 Characteristics of Pierce™ High-Capacity Ni-IMAC Magnetic Beads, EDTA Compatible.

Composition

Magnetization Ferrimagnetic with low remanence

Mean Diameter 30 µm

pH Tolerance 2–13

Reusability Up to 5 times

Binding Capacity ≥80 mg green fluorescent protein (GFP)/mL of settled beads

Chelator Stability Stable in buer containing 20 mM EDTA and 20 mM DTT

Magnetite-embedded agarose beads coupled to a novel, proprietary ligand loaded with nickel ions

Contents and storage

Product

Pierce™ High-Capacity Ni-IMAC Magnetic
Beads, EDTA Compatible

Cat. No. Amount Storage

A50588 1 mL of 25% slurry

A50589 5 mL of 25% slurry

Store at 4°C.

A50590 25 mL of 25% slurry

A50591 100 mL of 25% slurry

Important product information

• Do not centrifuge, dry, or freeze the Pierce™ High-Capacity Ni-IMAC Magnetic Beads, EDTA Compatible. Handling the beads in this
way will cause the beads to aggregate and lose binding capacity.

• Protein yield and purity are dependent upon the expression-level, conformation, and solubility characteristics of the recombinant
fusion protein; therefore, it is important to optimize these parameters. For best results, perform a small-scale test to estimate the
expression level and determine the solubility of each His-tagged protein.

For Research Use Only. Not for use in diagnostic procedures.

• Optimization of the lysis procedure is critical for maximizing protein yield. Some methods for protein extraction include using
commercially available detergent-based reagents, such as Thermo Scientific™ B-PER™ with Enzymes Bacterial Protein Extraction Kit
(Cat. No. 90078) and mechanical methods including freeze/thaw cycles, sonication or, French press.

• These instructions are eective for many types of samples; however, optimization may be required to further reduce nonspecific
binding. To optimize conditions, adjust the recommended imidazole concentration in the Equilibration, Wash, and Elution Buers.

• Concentration of proteins in the eluted fractions can be determined by using the Thermo Scientific™ Pierce™ Detergent Compatible
Bradford Assay Kit (Cat. No. 23246).

• When scaling up, use 2–3 volumes of Equilibration, Wash, and Elution Buers per volume of settled beads.

Materials required but not provided

Note: The buers listed below are recommendations. To decrease nonspecific binding and increase yield, adjustments to the imidazole

concentration may be required for specific proteins.

• Vary the imidazole concentration in the Elution Buer from 250 mM to 500 mM.

• Vary the imidazole concentration in the Equilibration Buer from 5 mM to 50 mM and in the Wash Buers from 10 mM to 50 mM.

• Purification of GFP from bacterial cell lysate is optimal with 10 mM imidazole in the Equilibration Buer and 20 mM imidazole in the
Wash Buer.

For native conditions, prepare the following buers:

• Equilibration Buer: 50mM monosodium phosphate, 300 mM sodium chloride, 10 mM imidazole in water; pH 8.0

• Wash Buer: 50 mM monosodium phosphate, 300 mM sodium chloride, 20 mM imidazole in water; pH 8.0

• Elution Buer: 50 mM monosodium phosphate, 300 mM sodium chloride, 500 mM imidazole in water; pH 8.0

For denaturing conditions, prepare the following buers:

• Equilibration Buer: 50 mM monosodium phosphate, 10 mM Tris base, 8 M urea in water; pH 8.0

• Wash Buer: 50 mM monosodium phosphate, 10 mM Tris base, 8 M urea in water; pH 6.3

• Elution Buer: 50 mM monosodium phosphate, 10 mM Tris base, 8 M urea in water; pH 4.5

For magnetic bead regeneration, prepare the following buers:

• 0.1 M NaOH, pH 13

• Neutralization Buer: 150 mM sodium chloride, 200 mM Na2HPO4; pH 7.0

• Storage Buer: 20% ethanol, 10 mM sodium acetate; pH 4.5

Manual purification of His-tagged proteins

Materials required but not provided

• 1.5 mL microcentrifuge tubes

• Sample containing His-tagged proteins

• Magnetic stand (e.g., Thermo Scientific™ MagnaBind™ Magnet for 6 × 1.5 mL microcentrifuge tubes, Cat. No. 21359)

Perform manual purification

Refer to “Materials required but not provided” on page 2 for composition of the recommended buers when using Native or Denaturing
conditions.

1. Place 40 µL (10 µL of settled beads) of Pierce™ High-Capacity Ni-IMAC Magnetic Beads, EDTA Compatible into a 1.5-mL
microcentrifuge tube.

2. Add 500 μL of Equilibration Buer and mix by vortexing. Place the tube on a magnetic microtube stand until the beads are separated
and discard the supernatant.

3. Pipet 500 μL of the clarified sample onto the equilibrated magnetic beads and incubate the sample/magnetic bead mixture at 4°C for
30 mins on an end-over-end shaker.

4. Place the tube on the magnetic stand until the beads separate and remove the supernatant. Optimize separation by briefly
centrifuging the sample to collect liquid from the lid before placing it on the magnetic separator.

2 Pierce

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High-Capacity Ni-IMAC Magnetic Beads, EDTA Compatible User Guide