Sep 16, 2024

Public workspaceMicro Volume Purification of His-Tagged Proteins with IMAC Ni-Charged Resin

DOI
dx.doi.org/10.17504/protocols.io.3byl49yejgo5/v1
  • Syon Schlecht1,2,
  • Emily Gunderson2,
  • Ruthie Fowler1,2,
  • Takara Aguilar3,2
  • 1Georgia Institute of Technology;
  • 2Empower College and Career Center;
  • 3University of Georgia
Syon Schlecht
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DOI: dx.doi.org/10.17504/protocols.io.3byl49yejgo5/v1
Protocol Citation: Syon Schlecht, Emily Gunderson, Ruthie Fowler, Takara Aguilar 2024. Micro Volume Purification of His-Tagged Proteins with IMAC Ni-Charged Resin. protocols.io https://dx.doi.org/10.17504/protocols.io.3byl49yejgo5/v1
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: September 09, 2024
Last Modified: September 16, 2024
Protocol Integer ID: 107141
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Abstract
This protocol describes a micro volume purification method for His-tagged proteins using IMAC with Ni-charged resin in a mini spin chromatography column. His-tagged proteins are selective bound to a Ni-charged resin due to the interaction between the polyhistidine tag and Ni²⁺ ions and eluted using imidazole-containing buffers. Desalting is the performed using SEC where salts and imidazole are fractionated, while larger molecules like the target protein are excluded. This results in the isolation of purified His-tagged proteins with a high yield and minimal contaminants, suitable for downstream applications.



Image Attribution
Created in BioRender. Schlecht, S. (2024) BioRender.com/d98d031
Protocol materials
ReagentMini Bio-Spin® Chromatography ColumnBio-Rad Laboratories
In 11 steps
ReagentMicro Bio-Spin® Column with Bio-Gel® P-6Bio-Rad Laboratories
In 6 steps
ReagentProfinity™ Ni-charged IMAC ResinBio-Rad LaboratoriesCatalog #1560131
Step 1
ReagentEquilibration Buffer
Step 5
ReagentWash Buffer
Step 9
ReagentElution Buffer
Step 10
Preparing and Equilibrating Column
Preparing and Equilibrating Column
6m
6m
Resuspend ReagentProfinity™ Ni-charged IMAC ResinBio-Rad LaboratoriesCatalog #1560131 and pipette Amount200 µL into a ReagentMini Bio-Spin® Chromatography ColumnBio-Rad Laboratories

Centrifuge theReagentMini Bio-Spin® Chromatography ColumnBio-Rad Laboratories at Centrifigation1000 x g, 00:02:00 and discard any collected buffer

2m
Wash the ReagentMini Bio-Spin® Chromatography ColumnBio-Rad Laboratories by adding Amount200 µL of distilled water

Centrifuge theReagentMini Bio-Spin® Chromatography ColumnBio-Rad Laboratories at Centrifigation1000 x g, 00:02:00 and discard any water collected

2m
Equilibriate the column by adding Amount200 µL of ReagentEquilibration Buffer to the ReagentMini Bio-Spin® Chromatography ColumnBio-Rad Laboratories
Note
The equilibration buffer is 20 mM sodium phosphate, 300 mM NaCl, and 5 mM imidazole.



Centrifuge theReagentMini Bio-Spin® Chromatography ColumnBio-Rad Laboratories at Centrifigation1000 x g, 00:02:00 and discard any buffer collected and cap the column

2m
Sample Binding and Elution of His-Proteins
Sample Binding and Elution of His-Proteins
20m
20m
From the soluble fraction of lysed cells containing the SampleHis-Sample add Amount600 µL to the ReagentMini Bio-Spin® Chromatography ColumnBio-Rad Laboratories and gently mix for Duration00:20:00

20m
Centrifuge theReagentMini Bio-Spin® Chromatography ColumnBio-Rad Laboratories at Centrifigation1000 x g, 00:02:00 and discard any flowthrough
2m
Add Amount600 µL of ReagentWash Buffer to the ReagentMini Bio-Spin® Chromatography ColumnBio-Rad Laboratories and centrifuge at Centrifigation1000 x g, 00:02:00 and discard any collected buffer
Note
The wash buffer is 20 mM sodium phosphate, 300 mM NaCl and 10 mM imidazole.



2m
Add Amount100 µL ReagentElution Buffer to the ReagentMini Bio-Spin® Chromatography ColumnBio-Rad Laboratories and centrifuge at Centrifigation1000 x g, 00:02:00 , and COLLECT the flowthrough, containing the SampleHis-Sample

Note
The elution buffer is 20 mM sodium phosphate, 300 mM NaCl, 250 mM imidazole, pH 8.0

Note
The ReagentMini Bio-Spin® Chromatography ColumnBio-Rad Laboratories can now be disposed of



2m
Critical
Desalting of His-Sample
Desalting of His-Sample
2m
2m
Snap the bottom and remove the top of a ReagentMicro Bio-Spin® Column with Bio-Gel® P-6Bio-Rad Laboratories and allow the packing buffer to drain for Duration00:05:00
Note
The ReagentMicro Bio-Spin® Column with Bio-Gel® P-6Bio-Rad Laboratories has a fractionation range of 1000 to 6000 Da, so any remaining salts or small proteins will be fractionated.



5m
Centrifuge theReagentMicro Bio-Spin® Column with Bio-Gel® P-6Bio-Rad Laboratories at Centrifigation1000 x g, 00:02:00 and discard any collected buffer

2m
Add the approx. Amount100 µL of the collected SampleHis-Sample to the ReagentMicro Bio-Spin® Column with Bio-Gel® P-6Bio-Rad Laboratories
Note
The maximum load volume of the column is Amount100 µL


Centrifuge the ReagentMicro Bio-Spin® Column with Bio-Gel® P-6Bio-Rad Laboratories at Centrifigation1000 x g, 00:05:00 and any excluded liquid is the purified SampleHis-Sample

5m
Repeat up to 3 times with the same ReagentMicro Bio-Spin® Column with Bio-Gel® P-6Bio-Rad Laboratories to obtain the desired SampleHis-Sample volume

Protocol references
Protein Expression and Purification Series.
https://www.bio-rad.com/webroot/web/pdf/lse/literature/pepsi_hr_1665067.pdf

Schlecht, S., Gunderson, E., Fowler, R., & Aguilar, T. (2024). Synthesis and Characterization of Naproxen-Salicylate Derivatives as Potential Dual-Targeted Inhibitors of Dihydrofolate Reductase. Advances in Biological Chemistry, 14(04), 87–102. https://doi.org/10.4236/abc.2024.144008

His-tagged Proteins – Production and Purification. ThermoFisher Scientific. https://www.thermofisher.com/us/en/home/life-science/protein-biology/protein-biology-learning-center/protein-biology-resource-library/pierce-protein-methods/his-tagged-proteins-production-purification.html