May 29, 2020
Version 1
NEBExpress Ni Spin Column Reaction Protocol (NEB #S1427) V.1
This protocol is a draft, published without a DOI.
- 1New England Biolabs
- FSU iGEM
External link: https://www.neb.com/protocols/2019/08/28/nebexpress-ni-spin-column-reaction-protocol-neb-s1427
Protocol Citation: New England Biolabs 2020. NEBExpress Ni Spin Column Reaction Protocol (NEB #S1427). protocols.io https://protocols.io/view/nebexpress-ni-spin-column-reaction-protocol-neb-s1-bfa3jign
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 20, 2020
Last Modified: May 29, 2020
Protocol Integer ID: 35899
Keywords: Ni Spin, purification, His-tagged, native, denaturing
Abstract
Ni resin can be used for the purification of His-tagged fusion proteins under native or denaturing conditions
- The binding capacity of NEBExpress® Ni Spin Columns is ≥ 1 mg per column. The binding capacity can 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.
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 Spin Columns – 25 columnsNew England BiolabsCatalog #S1427L
NEBExpressNi Spin Columns – 10 columnsNew England BiolabsCatalog #S1427S
Sodium Phosphate
NaCl
Imidazole, pH 7.4
H2O
Centrifuge
2ml microcentrifuge tubes
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 Spin Columns is ≥ 1 mg per column. The binding capacity can 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.
Buffer Preparation for Ni Spin Columns
Buffer Preparation for Ni Spin Columns
Prepare buffers for Ni Spin Columns as follows:
| Lysis/Binding Buffer: 20 mM sodium phosphate, 300 mM NaCl | Wash Buffer: 20 mM sodium phosphate, 300 mM NaCl, 5 mM Imidazole | Elution Buffer: 20 mM sodium phosphate, 300 mM NaCl, 500 mM Imidazole | ||
| 2X IMAC Buffer | 7.5 ml | 5.0 ml | 2.5 ml | |
| 2M Imidazole | - | 0.025 ml | 1.25 ml | |
| H2O | 7.5 ml | 5 ml | 1.25 ml | |
| Total | 15.0 ml | 10.0 ml | 5.0 ml |
Note
Notes:
- When the recommended protocol is followed, each isolation requires the following volumes of buffer: 0.75 mL Lysis/Binding Buffer , 0.75 mL Wash Buffer and 0.4 mL Elution Buffer . An excess volume of each concentrated buffer is provided for preparation of cell lysates or to optimize the concentration of imidazole in the washes.
- 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 10 mM (≥10 mM can reduce the isolated yield but may result in increased purity). imidazole concentration should be determined empirically.
- Refer to the Chemical Compatibility table prior to including other additives.
To prepare buffers for Ni Spin Columns under Native Conditions: bring all three buffers to a final pH of 7.4
To prepare buffers for Ni Spin Columns under Denaturing Conditions: bring all three buffers to a final concentration of 8 Molarity (M) Urea or 6 Molarity (M) Guanidine .
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 a 2 µl aliquot of the clarified lysate for SDS-PAGE analysis.
Note
Note: A standard isolation typically employs 0.5 ml of clarified lysate.
Column Preparation
Column Preparation
Remove the bottom tab of the column by twisting, loosen the top cap and place the column in the collection tube provided.
Centrifuge column at 800 x g, 00:01:00 to remove the storage buffer, discard the buffer.
Add 250 µL Lysis/Binding buffer to the column.
Centrifuge column at 800 x g, 00:01:00 , discard the Lysis/Binding buffer.
Place the column in a new 2 ml microcentrifuge tube.
Lysate Binding
Lysate Binding
Add up to 500 µL protein sample lysate to the column.
Note
Note: If sample volume is greater than 500 μl multiple applications can be performed; collect the flow through in separate microcentrifuge tubes.
Tap the column to mix the lysate with the resin and allow binding for 00:02:00 .
Note
Note: Binding of some His-tagged proteins can be increased with longer mixing times. Cap the column and seal the bottom using the plug provided. Mix end-over-end at 4 °C for desired time (typically 00:05:00 -00:15:00 ). Prolonged mixing may result in more non-specific binding.
Centrifuge column at 800 x g, 00:01:00 , reserve flow through.
Place the column in a new 2 ml microcentrifuge tube.
Column Wash
Column Wash
Add 250 µL wash buffer to the column and centrifuge at 800 x g, 00:01:00 .
Repeat wash step twice, collect each wash in a separate 2 ml microcentrifuge tube: Add 250 µL wash buffer to the column and centrifuge at 800 x g, 00:01:00 . (1/2)
Repeat wash step twice, collect each wash in a separate 2 ml microcentrifuge tube: Add 250 µL wash buffer to the column and centrifuge at 800 x g, 00:01:00 . (2/2)
Protein Elution
Protein Elution
Place column in a new 2 ml microcentrifuge tube.
Add 200 µL elution buffer to the column. Mix the resin with the elution buffer thoroughly.
Note
Note: Elution volume can be reduced to 100 µL if a more concentrated protein sample is desired, total target protein yield may be lower.
Centrifuge at 800 x g, 00:01:00 , save eluted sample.
Place column in a new 2 ml microcentrifuge tube and repeat elution step. Typically, >90% of the bound protein is eluted following the second elution.
Analyze the clarified cell lysate (load), flow through, washes and eluates by SDS-PAGE.