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 ecient 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 anity 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 buer 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 eective 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 Buers.
• 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 Buers per volume of settled beads.
Materials required but not provided
Note: The buers 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 Buer from 250 mM to 500 mM.
• Vary the imidazole concentration in the Equilibration Buer from 5 mM to 50 mM and in the Wash Buers from 10 mM to 50 mM.
• Purification of GFP from bacterial cell lysate is optimal with 10 mM imidazole in the Equilibration Buer and 20 mM imidazole in the
Wash Buer.
For native conditions, prepare the following buers:
• Equilibration Buer: 50mM monosodium phosphate, 300 mM sodium chloride, 10 mM imidazole in water; pH 8.0
• Wash Buer: 50 mM monosodium phosphate, 300 mM sodium chloride, 20 mM imidazole in water; pH 8.0
• Elution Buer: 50 mM monosodium phosphate, 300 mM sodium chloride, 500 mM imidazole in water; pH 8.0
For denaturing conditions, prepare the following buers:
• Equilibration Buer: 50 mM monosodium phosphate, 10 mM Tris base, 8 M urea in water; pH 8.0
• Wash Buer: 50 mM monosodium phosphate, 10 mM Tris base, 8 M urea in water; pH 6.3
• Elution Buer: 50 mM monosodium phosphate, 10 mM Tris base, 8 M urea in water; pH 4.5
For magnetic bead regeneration, prepare the following buers:
• 0.1 M NaOH, pH 13
• Neutralization Buer: 150 mM sodium chloride, 200 mM Na2HPO4; pH 7.0
• Storage Buer: 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 buers 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 Buer 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
™
High-Capacity Ni-IMAC Magnetic Beads, EDTA Compatible User Guide
5. Remove the tube from the magnet. Add 500 μL of Wash Buer and mix by vortexing. Place the tube again on the magnetic stand
and allow the beads to separate. Remove the supernatant.
6. Repeat step 5 twice.
7. Elute the His-tagged protein using 100 μL of Elution Buer.
Note: Depending on the protein expression rate and desired protein concentration, the elution volume can be adjusted from 25 µL to
500 μL.
8. Repeat step 7 three times. Collect each elution fraction in a separate tube and determine the protein concentration of each fraction.
9. Analyze all fractions by SDS-PAGE.
Note: Do not boil membrane proteins. Instead, incubate the sample at 46°C for 30 mins in preparation for SDS-PAGE analysis.
Regenerate the magnetic beads
1. After completion of purification, add 500 µL of deionized water to 10 µL of settled magnetic beads, then place the tube on the
magnetic stand and allow beads to separate. Remove the supernatant.
2. Repeat step 1.
3. Add 500 µL of NaOH to the magnetic beads and incubate for 10 mins. Place tube on the magnetic stand and allow beads to
separate. Remove the supernatant.
4. Add 500 µL of deionized water to the magnetic beads, then place the tube on the magnetic stand and allow beads to separate.
Remove the supernatant.
5. Add 500 µL of Neutralization Buer (150 mM sodium chloride; 200 mM Na2HPO4, pH 7.0) to magnetic beads, then place the tube on
the magnetic stand and allow the beads to separate. Remove the supernatant.
6. Add 500 µL of deionized water to the magnetic beads, then place the tube on the magnetic stand and allow beads to separate.
Remove the supernatant.
7. Repeat step 6 and proceed to purification.
8. For long-term storage, add 500 µL of Storage Buer and store at 4°C.
Automated purification of His-tagged proteins
Materials required but not provided
• KingFisher™ Flex Magnetic Particle Processor with 96 Deep-Well Head (Cat. No. 5400630) or KingFisher™ Duo Prime Purification
System (Cat. No. 5400110)
• Thermo Scientific™ Microtiter Deep Well 96 Plate, V-bottom, polypropylene (100–1,000 µL, Cat. No. 95040450)
• KingFisher™ 96 tip comb for deep-well magnets (Cat. No. 97002534)
Pierce™ High-Capacity Ni-IMAC Magnetic Beads, EDTA Compatible User Guide 3
Instrument preparation and plate setup
Note: The following protocol is designed for use with the KingFisher™ Flex instrument. The protocol can be modified according to your
needs using the BindIt™ Software provided with the instrument.
1. Download the "Ni-IMAC" protocol from the Thermo Fisher Scientific website at https://www.thermofisher.com/us/en/home/life-
science/dna-rna-purification-analysis/automated-purification-extraction/automated-protocols-software.html?open=protein
into the BindIt™ Software on an external computer.
2. Transfer the protocol to the KingFisher™ Flex instrument from an external computer. See the BindIt™ Software User Manual for
detailed instructions on importing protocols.
3. Set up plates according to Table 2.
Table 2 Pipetting instructions for the His-tagged Protein Purification protocol using the Thermo Scientific™ Microtiter Deep Well
96 Plates.
Plate #
1 Beads
2 Bead Equilibration Equilibration Buer 500 µL 30 secs/Medium
3 Bind
4 Wash 1 Wash Buer 500 µL 30 secs/Medium
5 Wash 2 Wash Buer 500 µL 30 secs/Medium
6 Wash 3 Wash Buer 500 µL 30 secs/Medium
7 Elution 1 Elution Buer 200 µL 10 mins/Medium
8 Tip Plate
Plate Name Content Volume Time/Speed
Beads 40 µL
Equilibration Buer 500 µL
Protein in Equilibration
Buer
KingFisher™ 96 tip comb
for DW magnets
500 µL 30 mins/Medium
— 10 secs/Fast
15 secs
Perform automated purification
1. Select the protocol using the arrow keys on the instrument keypad and press Start. See the KingFisher™ Flex instrument user guide
for detailed information.
2. Slide open the door of the instrument's protective cover.
3. Load plates into the instrument according to the protocol requests, placing each plate in the same orientation. Confirm each action
by pressing Start.
4. After sample processing, remove the plates as instructed by the instrument’s display. Press Start after each plate. Press Stop after
removal of plates.
Note:
If fewer than 96 wells are used, fill the same wells in each plate. For example, if using wells A1 through A12, use these same wells
·
in all plates.
Combine the Tip Comb with a Deep Well 96 Plate. See the instrument user manual for detailed instructions.
·
A minimum volume of 100 mL is required for ecient elution of bound protein
·
Troubleshooting
Observation
Low protein yield. Poor expression of soluble protein. Optimize expression conditions.
His-tagged protein formed inclusion
bodies.
4 Pierce
Possible cause Recommended action
Alter growth conditions to minimize inclusion body formation and
maximize soluble protein yield. Alternatively, solubilize inclusion bodies
and perform the purification with a compatible denaturant (e.g.,
Thermo Scientific™ Inclusion Body Solubilization Reagent, Cat. No.
78115) using optimized denaturing conditions. See “Materials required
but not provided” on page 2 for recommended denaturing buers.
™
High-Capacity Ni-IMAC Magnetic Beads, EDTA Compatible User Guide
Observation Possible cause Recommended action
Low protein yield.
(continued)
Insucient cell lysis and extraction. Optimize the cell lysis protocol.
Fusion protein did not bind to the
magnetic beads.
Verify the sequence.
Perform an ELISA or western blot using an antibody against the Histagged protein to ensure the His-tagged protein is present. Decrease
imidazole concentration in the Equilibration and/or Wash Buer.
Poor protein purity. Insucient washing. Wash beads a minimum of two additional times.
Adjust imidazole concentration of the Equilibration and/or Wash
Buer.
Related products
Product
Catalog Number
Pierce™ High-Capacity Ni-IMAC Resin, EDTA Compatible A50584-7
B-PER™ Bacterial Protein Extraction Reagent with Enzymes 90078
Expi293™ Expression System Kit A14635
ExpiCHO™ Expression System Kit A29133
ExpiSf™ Expression System Starter Kit A38841
Pierce™ Detergent Compatible Bradford Assay Kit 23246
Pierce™ 660nm Protein Assay Kit 22662
Pierce™ DTT (Dithiothreitol), No-Weigh™ Format A39255
UltraPure™ 0.5M EDTA, pH 8.0 15575020
MagnaBind™ Magnet for 6 × 1.5mL microcentrifuge tubes 21359
Pierce™ Protease and Phosphatase Inhibitor Tablets Multiple products available at thermofisher.com
Halt™ Protease Inhibitor Cocktail Multiple products available at thermofisher.com
Pierce™ Concentrators Multiple products available at thermofisher.com
Novex™ WedgeWell™ Tris-Glycine Mini Gels Multiple products available at thermofisher.com
Zeba™ Spin Desalting Columns Multiple products available at thermofisher.com
Slide-A-Lyzer™ Dialysis Cassettes Multiple products available at thermofisher.com
Limited product warranty
Life Technologies Corporation and/or its aliate(s) warrant their products as set forth in the Life Technologies' General Terms and
Conditions of Sale at www.thermofisher.com/us/en/home/global/terms-and-conditions.html. If you have any questions, please
contact Life Technologies at www.thermofisher.com/support.
Thermo Fisher
For descriptions of symbols on product labels or product documents, go to thermofisher.com/symbols-definition.
The information in this guide is subject to change without notice.
DISCLAIMER: TO THE EXTENT ALLOWED BY LAW, THERMO FISHER SCIENTIFIC INC. AND/OR ITS AFFILIATE(S) WILL NOT BE LIABLE FOR SPECIAL, INCIDENTAL, INDIRECT,
PUNITIVE, MULTIPLE, OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT, INCLUDING YOUR USE OF IT.
Revision history: Pub. No. MAN0024861
Important Licensing Information: These products may be covered by one or more Limited Use Label Licenses. By use of these products, you accept the terms and conditions of all
applicable Limited Use Label Licenses.
©2021 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified.
thermofisher.com/support | thermofisher.com/askaquestion
thermofisher.com
15 February 2021
Scientific | 3747 N. Meridian Road | Rockford, Illinois 61101 USA
Revision Date Description
A.0 15 February 2021 New manual.
Loading...