Thermo Fisher Scientific A50584 User Manual

USER GUIDE

Pierce™ High-Capacity Ni-IMAC Resin, EDTA Compatible

Catalog Numbers A50584

Pub. No. MAN0024862 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 Resin, EDTA Compatible enables ecient purification of recombinant polyhistidine-tagged proteins
from a soluble protein extract or mammalian cell culture supernatant. This resin consists of 6% crosslinked agarose beads coupled to a
novel proprietary ligand loaded with nickel ions. The novel high-anity chelator of the resin enables high binding capacity of His-tagged
proteins in the presence of chemical additives such as 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 resins. The resin is compatible with native or
denaturing conditions and can be used in multiple formats, including conventional gravity-flow chromatography, spin column, and FPLC.

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

Binding Capacity

Resin Ferrimagnetic with low remanence

pH Tolerance 2–13

Supplied 50% slurry in 20% ethanol

Reusable Up to 10 times

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 Resin, EDTA
Compatible

Cat. No. Amount Storage

A50584 20 mL of 50% slurry

A50585 100 mL of 50% slurry

Store at 4°C.

A50586 500 mL of 50% slurry

A50587 1,000 mL of 50% slurry

Important product information

• 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 before attemtping a large-scale purification. For best results,
perform a small-scale test to estimate the expression level and determine the solubility of each His-tagged protein.

• 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. Add protease inhibitors Thermo

Scientific™ Halt™ Protease and Phosphatase Inhibitor Cocktail (100X) (Cat. No. 78444) to protect proteins from degradation.

• Overexpressed proteins can be sequestered in inclusion bodies. Inclusion bodies of His-tagged proteins can be solubilized in 8 M
urea, 6 M guanidine, or Thermo Scientific™ Inclusion Body Solubilization Reagent (Cat. No. 78115) and purified with the Ni-IMAC
resin, but a denaturant must be added to buers, so the protein remains soluble throughout the procedure.

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

• 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) or Pierce™ 660nm Protein Assay Kit (Cat. No. 22662).

• For liquid chromatography applications, use highly pure buer components and ultrapure water. Use low-absorbance imidazole
(Fisher Scientific, Cat. No. BP 305-50) to avoid UV interference. Degas or filter buers through a 0.45 μm filter before use.

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.

For native conditions, prepare the following buers:

• Equilibration Buer: 50 mM 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: 100 mM monosodium phosphate, 10 mM Tris base, 8 M urea in water; pH 8.0

• Wash Buer: 100 mM monosodium phosphate, 10 mM Tris base, 8 M urea in water; pH 6.3

• Elution Buer: 100 mM monosodium phosphate, 10 mM Tris base, 8 M urea in water; pH 4.5

For resin 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 6.5

Purify His-tagged proteins by Batch Method (Native and Denaturing conditions)

The Pierce™ High-Capacity Ni-IMAC Resin, EDTA Compatible allows for purification strategy customization. Purification conditions can be
scaled as needed. The procedure may be performed at room temperature or at 4°C. Refer to “Materials required but not provided” on
page 2 for corresponding buer formulations under Native and Denaturing Conditions.

1. Resuspend the resin by inverting the bottle until the suspension is homogeneous. Transfer 1 mL of the 50% suspension
(corresponding to 500 μL bed volume) to a 15 mL conical centrifuge tube. Allow the resin to settle by gravity and remove the
supernatant. Alternatively, resin equilibration can be performed directly in a disposable gravity flow column (see thermofisher.com
for Pierce™ Disposable Columns).

2. Add 2.5 mL of Equilibration Buer and gently resuspend the slurry to equilibrate the resin. Allow the resin to settle by gravity and
remove 2 mL of supernatant.

3. Add 10 mL of clarified sample to the equilibrated resin and incubate at 4°C for 1 hour on an end-over-end shaker. Alternatively,
batch binding can be performed directly in a gravity flow column with closed bottom and top outlets.

4. Transfer the binding suspension to a disposable gravity flow column with a capped bottom outlet. Use Equilibration Buer to rinse
the centrifuge tube and remove resin adhered to the wall.

5. Remove the bottom cap of the column and collect the flowthrough.

6. Wash the column with 5 mL of Wash Buer. Repeat the washing step at least 3 times.

7. Elute the His-tagged protein 5 times using 0.5 mL of Elution Buer. Collect each eluate in a separate tube and determine the protein

concentration of each fraction. (Optional) To increase protein yields, incubate the resin for 15 mins in Elution Buer before collecting
the eluate.

Note: Monitor protein elution by measuring the absorbance of the fractions at 280 nm. The eluted protein can be directly analyzed by
SDS-PAGE. To remove excess imidazole for downstream applications, use gel filtration or dialysis (e.g., Thermo Scientific™ Zeba
Spin Desalting Columns or Slide-A-Lyzer™ Dialysis Cassettes (see “Related products” on page 4).

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8. Analyze all fractions by SDS-PAGE.

Note: Do not boil membrane proteins. Instead, incubate samples at 46°C for 30 mins in preparation for SDS-PAGE analysis.

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High-Capacity Ni-IMAC Resin, EDTA Compatible User Guide