Oct 22, 2021

Public workspaceProtein purification

This protocol is a draft, published without a DOI.
  • 1iGEM Gifu
  • iGEM Gifu
Yuichiroh Ikagawa
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Protocol Citation: Yuichiroh Ikagawa 2021. Protein purification. protocols.io https://protocols.io/view/protein-purification-bzcsp2we
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: In development
We are still developing and optimizing this protocol
Created: October 21, 2021
Last Modified: October 22, 2021
Protocol Integer ID: 54386
Abstract
The extraction method is based on the paper by Janice S. Chen et al. The cultured E. coli is collected by centrifugation and then disrupted by sonication. The extracted solution is purified by affinity chromatography using a Ni-NTA column with an x10 His tag attached to Cas12a. Clontech's His60 Ni Gravity Column Purification Kit is used for purification, and the experimental method is based on the kit protocol.
Materials
Reagents
PBS
Lysis buffer (50 mM Tris-HCl, pH 7.5, 500 mM NaCl, 5% (v/v) glycerol, 0.25 mg/ml lysozyme)
His60 Ni Gravity Column Purification Kit (Clontech Laboratories, Inc.)
  • Equilibrium buffer (50 mM sodium phosphate, 300 mM sodium chloride, 20 mM imidazole; pH 7.4)
  • Wash buffer (Elution buffer: 710 μl, Equilibration Buffer: 9.29 ml)
  • Elution buffer (50 mM sodium phosphate, 300 mM sodium chloride, 300 mM imidazole; pH 7.4 )

Equipment
Sonicator
His60 Ni Gravity Column (Clontech Laboratories, Inc.)
extraction
extraction
Transfer 50 ml of culture medium to a 50 ml tube.
Collect the pellet by centrifugation at 5,000 g for 5 minutesTemperature4 °C

Resuspend the pellet with 2 ml PBS.
Dispense the culture medium into 1.5 ml tubes.
Centrifuge at 15,000 g for 30 minutes.
Resuspend 500 μl Lysis buffer.
Disrupt the cell by sonication (x10 sonication: 10 seconds/incubation: 10 seconds) on ice.
protein purification
protein purification

Fully suspend the matrix before opening the column.Temperature4 °C

Carefully remove the bottom stopper.
Add 1ml of Equilibration buffer to the His60 Ni gravity column 10 times. Temperature4 °C

Attach the bottom stopper.
Add cell extraction sample to the column.Temperature4 °C

Carefully connect the top stopper to the top of the column.
Slowly Inverting the column for 1 hour.Temperature4 °C

Install the column in a vertical position and let the resin settle at the bottom of the column. Temperature4 °C

Carefully remove the top stopper.
Remove the bottom stopper and collect 1 ml fraction each into 1.5 ml tubes.Temperature4 °C

Add 1 ml Equilibration buffer to the column 10 times.Temperature4 °C

Add 1 ml Wash Buffer to the column 10 times.Temperature4 °C

Add Elution Buffer to the column and collect 1 ml fractions.Temperature4 °C

Measure the absorbance 280 nm of the fractions by nanodrop.
Added 1 ml Equilibration Buffer to the column 20 times. Temperature4 °C

Wash the column with 1 ml of water 5 timesTemperature4 °C

Attach the bottom stopper.
Add 20% ethanol to the column for storage.Temperature4 °C

Attach top stopper and storage.Temperature4 °C