Purification, detection, and characterization of proteins

lecture 6

How do proteins vary? (what characteristics?)

size, charge, hydrophobicity

how does separation by chromatography work?

separation of components is based on their different interactions with the immobile material in the column

the mobile liquids move at rate which depends on their interactions with the solid phase

how does gel filtration chromatography work?

what is based on?

separation is based on shape and size

the solid phase beads have pores in which the small proteins diffuse through, whereas the larger proteins are free to just move through the column first

then a buffer is added to disrupt the interactions between the gel beads and the small proteins allowing the small proteins to flow through

How does ion chromatography work and what is it bases on?

Separation based on electric charge

the gel beads in the column are positively charged so they attract the negatively charged proteins while the positive ones flow freely

then NaCl is added disrupting the interaction allowing negatively charged ions to come through as well as the Na - this can be removed through other methods

How does antibody affinity chromatography work and what is it based on?

Separation based on antibody recognition

the protein that is recognized by the antibody will stick to the antibody

then the pH can be lowered to release to get the target protein out

How does separation of proteins by electrophoresis work (PAGE) and what is it based on?

It is based on charge of protein and mass ratio

the proteins are run through a gel which contains an electric field 

the more negatively charged proteins will move further and the smaller proteins will move further

how does SDS page work and what is it based on?

separation based on size

proteins are denatured and coated in SDS which is very negative, making sure that their migration is more based on size rather than charge 

how does immunoblot work?

the next step of SDS page where we can visualize results 

proteins in SDS page are transferred to a membrane are exposed → antibody 1 is used to detect the protein of interest → coated with protein 2 (using fluorescence to detect the primary antibody)

darker and denser = more abundant protein

How does Co-immunoprecipitation work and what is it used for?

used to identify protein-protein interactions in a cell by using an antibody that is specific for one protein of the complex

example: GR and PPARa

What does immunofluorescence do?

identifies cellular localization of proteins 

1. cells killed using solvent 

2. immobilize on microscope slide

3. antibodies used to detect specific proteins and fluorescently labeled antibodies used to mark them at microscope  

How does labeling a protein with GFP work?

GFP is a fluorescent marker

modifying the gene at the genetic level

GFP can be used by fusing it to the C or N terminus and depends on the protein in question