May 18, 2020
Version 1
NEBExpress Ni Resin Batch Binding Typical Protocol (NEB #S1428) V.1
- 1New England Biolabs
- New England Biolabs (NEB)Tech. support phone: +1(800)632-7799 email: info@neb.com
Protocol Citation: New England Biolabs 2020. NEBExpress Ni Resin Batch Binding Typical Protocol (NEB #S1428). protocols.io https://dx.doi.org/10.17504/protocols.io.bfd5ji86
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: April 21, 2020
Last Modified: May 18, 2020
Protocol Integer ID: 35997
Keywords: Ni , resin, Ni resin, His-tagged, native, denaturing,
Abstract
NEBExpress® Ni Resin is an affinity matrix for the isolation and purification of polyhistidine-tagged (His-tagged) fusion proteins. It is intended for use in gravity or pressure flow columns, and batch purifications. NEBExpress Ni Resin is comprised of a highly uniform and stable chemical-tolerant resin, pre-charged with nickel ions on the matrix surface. It is resistant to a wide range of chemicals, including NaOH, EDTA, DTT and β-Mercaptoethanol.
- Purification of ≥10 mg His-tagged protein per 1 ml of resin
- Intended for use in gravity or pressure flow columns, and batch purifications
- High specific binding of His-tagged proteins yielding purities of >95%
- Strong nickel ion binding provides excellent resistance to EDTA and reducing agents. Compatible with commercially available detergent-based cell lysis reagents
- Isolation and purification of His-tagged fusion proteins under native or denaturing conditions
Guidelines
Chemical Compatibility
| Reagent | Tolerance (up to) | |
| EDTA | 10 mM*, 100 mM** | |
| DTT | 5 mM | |
| b-mercaptoethanol | 20 mM | |
| TCEP | 5 mM | |
| Triton X-100 | 2 % | |
| Tween 20 | 2 % | |
| NP-40 | 2 % | |
| Cholate | 2 % | |
| CHAPS | 1 % | |
| Tris-HCl, HEPES, MOPS | 100 mM | |
| Urea | 8 M | |
| Guanidine-HCl | 6 M |
* If reagents contain 10 mM EDTA, do not mix the sample and the resin for more than 24 hours before washing and eluting.
** If reagents contain 100 mM EDTA, do not mix the sample and the resin for more than 2 hours before washing and eluting.
Materials
MATERIALS
NEBExpressNi Resin – 25 mlNew England BiolabsCatalog #S1428S
Recommended Buffers
Lysis/Binding Buffer: 20 mM sodium phosphate, 300 mM NaCl, pH 7.4
Wash Buffer: 20 mM sodium phosphate, 300 mM NaCl, 5 mM Imidazole, pH 7.4
Elution Buffer: 20 mM sodium phosphate, 300 mM NaCl, 500mM Imidazole, pH 7.4
Safety warnings
Please refer to the Safety Data Sheets (SDS) for health and environmental hazards.
Before start
Ni resin can be used for the purification of His-tagged fusion proteins under native or denaturing conditions
·The binding capacity of NEBExpress® Ni Resin is ≥ 10 mg/ml. The binding capacity will vary depending on the size of the target protein, binding conditions and the accessibility of the His-tag. An exact protocol may need to be optimized by the user.
·It is recommended to estimate the expression level of the His-tagged protein of interest by first running a sample on an SDS-PAGE gel.
Purchase or prepare recommended buffers:
Recommended Buffers
Lysis/Binding Buffer: 20 mM sodium phosphate, 300 mM NaCl, pH 7.4
Wash Buffer: 20 mM sodium phosphate, 300 mM NaCl, 5 mM Imidazole, pH 7.4
Elution Buffer: 20 mM sodium phosphate, 300 mM NaCl, 500mM Imidazole, pH 7.4
Preparation of Buffers under Denaturing Conditions
Preparation of Buffers under Denaturing Conditions
If continuing with Denaturing conditions: Bring all three buffers (Lysis/Binding, Wash and Elution Buffers) to a final concentration of 8 Molarity (M) Urea or 6 Molarity (M) Guanidine .
Note
Notes:
1.Crude lysate should be prepared with a lysis buffer without imidazole. To further minimize contaminants in the eluate, the concentration of imidazole in the wash buffer can be increased to 15 mM (≥15 mM can reduce the isolated yield but may result in increased purity). Imidazole concentration should be determined empirically.
2. Refer to the Chemical Compatibility table prior to including other additives.
Sample Preparation
Sample Preparation
Harvest cells by centrifugation at 4000 x g, 00:15:00 , store the pellet at -20 °C or process immediately.
Note
Note: it is recommended to pre-weigh the vessel prior to addition of cell suspension, in order to determine the mass of cell pellet used.
Resuspend cell pellet in Lysis buffer and lyse using method of choice (use approximately 5 mL lysis buffer per 1 g cell paste ).
Note
Note: Cells can be lysed by standard methods including sonication, repeated freeze-thaw cycles, French press, etc. Other commercially available lysis reagents can also be used, following manufacturer’s instructions. It is recommended that imidazole be omitted from any lysis buffer.
Centrifuge sample at 12000 x g, 00:15:00 to pellet cellular debris. Remove the clarified protein lysate supernatant and transfer to a new microcentrifuge tube on ice, retain an aliquot of the clarified lysate for SDS-PAGE analysis.
Resin Preparation
Resin Preparation
Gently shake the Ni Resin bottle to completely resuspend the slurry and transfer desired amount to a tube.
Settle the resin by centrifugation at 500 x g, 00:05:00 , remove the supernatant.
Equilibrate the resin by adding 5 column volumes (CV) of Lysis/Binding buffer, shake gently to mix.
Settle the resin by centrifugation at 500 x g, 00:05:00 , remove the supernatant.
Repeat equilibration steps once:
Settle the resin by centrifugation at 500 x g, 00:05:00 , remove the supernatant.
Equilibrate the resin by adding 5 column volumes (CV) of Lysis/Binding buffer, shake gently to mix.
Settle the resin by centrifugation at 500 x g, 00:05:00 , remove the supernatant.
Batch Binding
Batch Binding
Add the protein sample lysate to the equilibrated resin. Mix gently by shaking at 200 rpm on a rotary shaker for ≥ 00:15:00 .
Note
Note: Binding of some His-tagged proteins can be increased with longer incubation times. Resin should be placed at4 °C when extending the binding time to maintain activity of the target protein. Prolonged incubation may result in more non-specific binding.
Pour the lysate/resin mixture into an empty and closed gravity column.
Open the column and collect the flow through in a clean tube. Reserve for SDS-PAGE analysis.
Column Wash
Column Wash
Add ≥ 10 CV of Wash buffer to the column, allow buffer to flow through. Flow through can be monitored with absorbance at 280 nm to ensure complete removal of unbound lysate effluent.
Collect flow through in fractions and reserve for SDS-PAGE analysis.
Protein Elution
Protein Elution
Elute fusion protein with Elution buffer.
Note
Note: it is recommended to use a minimum of 3 CV of elution buffer to elute the target protein. Optimal amounts may need to be determined empirically for different substrates.
Collect flow through in fractions and monitor protein elution by measuring the absorbance at 280 nm or by SDS-PAGE.
Analyze the protein sample lysate (load), flow through, washes and eluates by SDS-PAGE.
Note
Note: To remove imidazole for downstream applications use gel filtration (e.g., Zeba Spin Desalting Columns) or dialysis (e.g. slide-A-Lyzer Dialysis Cassettes)
Guidelines for Ni Resin Cleaning
Guidelines for Ni Resin Cleaning
Note
When using a gravity column, the flow rate should be determined prior to cleaning to ensure an accurate contact time with the cleaning agent.
To remove lipoproteins, lipids and proteins strongly bound by hydrophobic interaction follow one of these three methods:
- Add 1 Molarity (M) NaOH to the column and allow a contact time of 01:00:00 to 02:00:00 , followed by 10 CV of Lysis/Binding Buffer.
- Add 30% isopropanol to the column and allow a contact time of 00:15:00 to 00:20:00 , followed by 10 CV of water.
- Add 0.1 – 0.5% non-ionic detergent in 0.1 Molarity (M) acetic acid to the column and allow a contact time of 01:00:00 to 02:00:00 , followed by 10 CV of water.
To remove proteins bound by ionic interactions
- Add 1.5 Molarity (M) NaCl to the column and allow a contact time of 00:10:00 to 00:15:00 . followed by 10 CV of water.
Storage
Storage
For long-term storage, NEBExpress Ni Resin should be stored in 20% ethanol at 2 °C to 8 °C ..