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Protein Expression and Purification Core FacilityProtocols

Full List of Protocols

1. Cloning

Preparation of oligo solutions
PCR reactions
Digestion of insert DNA
Digestion and dephosphorylation of vector DNA
Ligation of DNA fragments with sticky ends
Ligation of DNA fragments with blunt ends
Ligation independent cloning (LIC)
Protocol for preparing electrocompetent E. coli
Protocol for preparing heat shock competent E. coli
Transformation of competent E. coli with vector DNA

1.1. Cloning by homologous recombination

Creating a GATEWAY Entry Clone via the BP Reaction
Creating a GATEWAY Expression Clone via the LR Reaction
One tube protocol to create a GATEWAY Expression Clone
2-Step GATEWAY PCR experiments

2. Expression

2.1. Plasmid Stability

Plasmid Stabilty Test

2.2. Protein Labelling

N15 labelling
N15 and C13 labelling
Seleno-methionine labelling

2.3. Protein Toxicity

"Plating" method for culture inoculation

3. Purification

3.1. Extraction and Clarification

Preparation of cell lysates from E. coli by sonication
Preparation of cell lysates from E. coli using a French Press
Preparation of cell lysates from E. coli by enzymatic lysis
Preparation of cell lysates from yeast using a French press
Preparation of cell lysates from yeast using glass beads vortexing
Preparation of cell lysates from yeast by enzymatic digestion of the cell wall
Solubilisation and renaturation of proteins from inclusion bodies

3.2. Purification

His-tag protein purification using Ni-NTA magnetic beads

4. Characterisation

4.1. Analytical Ultracentrifugation

Cell assembly
Equilibrium run
Velocity run
Data analysis

4.2. Chromatography

Calibration of Superose 12 HR 10/30
Calibration of Superdex 75 HR 10/30
Calibration of HiLoad 16/60 Superdex 75 prep grade
Calibration of HiLoad 26/60 Superdex 75 prep grade
Calibration of HiLoad 26/60 Superdex 200 prep grade