Protein Expression and Purification Series - Bio-Rad

Protein Expression and Purification Series
Advance Preparation for Chromatography Laboratory
In this Chromatography activity, affinity chromatography will be used to isolate the GST-DHFR-His
protein. The eluted GST-DHFR-His protein will subsequently be exchanged into a buffer with no imidazole
using size exclusion/desalting chromatography in preparation for SDS-PAGE analysis, quantitation, and
enzymatic activity analysis.
Protein Purification by Centrifugation Laboratory Checklist
Components from Protein Expression
and Purification Series Where Provided (✔)
Imidazole stock solution Growth and Expression Module ❒
10x PBS Growth and Expression Module ❒
Microcentrifuge tubes, 2 ml Growth and Expression Module ❒
Screwcap microcentrifuge tubes, 1.5 ml SDS-PAGE Electrophoresis Module ❒
Profinity IMAC Ni-charged resin Centrifugation Purification Module ❒
Empty Micro Bio-Spin columns Centrifugation Purification Module ❒
Desalting columns Centrifugation Purification Module ❒
Microcentrifuge collection tubes Centrifugation Purification Module ❒
Lysis buffer 2/Wash buffer Prepared for Cell Lysis Laboratory ❒
Required Accessories (Not Provided) Quantity (✔)
Microcentrifuge with variable speed setting >16,000 x g 1 ❒
Distilled water 20 ml ❒
2–20 µl adjustable-volume micropipets and tips 12 ❒
20–200 µl adjustable-volume micropipets and tips 12 ❒
100–1,000 µl adjustable-volume micropipets and tips 12 ❒
Tube roller or rocker platform 1 ❒
Marking pens 12 ❒
UV spectrophotometer 1 ❒
trUView cuvettes (or UV compatible cuvettes) 12 ❒
Tasks to Perform Prior to the Lab
1. Equilibration buffer: To prepare 20 ml of Equilibration buffer, combine 15.6 ml of distilled water, 4 ml
of 10x PBS, and 0.4 ml of imidazole stock solution, and mix thoroughly. Equilibration buffer should be
stored at 4°C and is good for one month. The Equilibration buffer is 20 mM sodium phosphate, 300
mM NaCl, and 5 mM imidazole.
Aliquot 500 µl of Equilibration buffer into a 2 ml microcentrifuge tube for each workstation.
2. Lysis buffer 2/Wash buffer: Prepared for Cell Lysis laboratory activities. Aliquot 600 µl of Lysis buffer
2/Wash buffer into a 2 ml microcentrifuge tube for each workstation.
3. Elution buffer: The imidazole stock solution will be used as the Elution buffer. Elution buffer should be
stored at 4°C and is good until the labeled expiration date. Elution buffer is 20 mM sodium phosphate,
300 mM NaCl, 250 mM imidazole, pH 8.0.
Aliquot 400 µl of Elution buffer into a 2 ml microcentrifuge tube for each workstation.
4. Aliquot 250 µl of Profinity IMAC Ni-charged resin slurry into a 2 ml microcentrifuge tube for each
workstation.
Chapter 3A: Advance Preparation
for Centrifugation Protocols
65
CHAPTER 3A
ADVANCE PREP
CENTRIFUGATION PROCES