Purification of proteins (6)

Basis of protein purification

Modify bacteria for its genome to overexpress a protein : extracted from cell and removing big cellular debris afterwards

Name of this method (size and shape)

Gel filtration chromatography (GFC)

Solid phase of GFC

Gel beads with pores of molecular dimensions where small proteins can enter and exit freely

Large proteins in GFC

Cannot enter solid phase so they migrate faster and are eluted earlier

Name of this method (electric charge)

Ion-exchange chromatography (IEC)

Solid phase of IEC

Positively charged bead

Behaviour of mobile phase (IEC)

Protein will bind to the solid phase having the opposite charge and are displaced (ion exchange) with a NaCl solution

Name of this method

Antibody affinity chromatography (AAC)

Solid phase of AAC

Covalently coupled to an antibody specific to a protein

Separation of antibody from the protein

Lowering the pH with a buffer solution

Principle of how electrophoresis works

Speed of migration is determined by charge/mass ratio + shape

Advantage of SDS-PAGE

Proteins only migrate depending of their size because charge (SDS coat) proportionate to their length and unfolded

Immunoblot

1. Proteins in SDS-PAGE gel transferred to membrane

2. Primary + Secondary = fluorescence

Co-immunoprecipitation

To co-purify other proteins that interact with it : allows to identify a protein in a protein complex

Ex of co-immunoprecipitation

Testing the interaction between GR and PPARa