Sept 20 Purification, Detection and Characterization of Proteins II Flashcards
what is chromatography?
separation of components based on their differential interaction with an immobile (=solid) material
how does the mobile (liquid) phase move?
it moves continuously past the solid phase
what does the rate of the protein moving up the solid depend on?
the rate depends on how much they interact with the solid phase
How does gel filtration chromatography work?
gel filtration separates proteins based on size
the solid phase gel beads have pores of molecular dimensions
the mobile phase freely enters and exits the beads and flows in between the beads
small proteins can enter and exit the beads through the pores
large proteins cannot enter: they don’t “waste time” within the beads, so they run ahead of the small proteins
the smaller the mass, the longer the protein spends trapped on the beads, the longer it takes to traverse the column
How does Ion-exchange chromatography work?
the separation is based on electric charge
protein molecules with a net electric charge will bind to immobile phase having the opposite charge
electrostatically bound proteins can be released by flowing a salt solution through the column
the displacement of the protein by Na+ or Cl- is “ion
what is an antibody and what does it recognize?
an antibody is a protein that recognises, by highly specific binding, a molecular target, called an epitope, present on the antigen molecule against which the antibody was raised
each antibody recognises a single epitope
how can antibodies be used when separating proteins?
antibodies can be raised against proteins
if the epitope recognised by the antibody is unique to the particular protein used as an antigen, then the antibody will recognise, and bind to only that individual protein, even in a complex mixture of proteins
How does antibody-affinity chromatography work?
antibodies specific for any particular protein can be covalently couples to the solid phase
if a complex protein mixture is flowed through the column, only the protein to which the antibody binds will be retained
all the other proteins can be washed away and then the target protein can be released from the antibody by lowering the pH
what are primary and secondary antibodies?
the primary antibodies recognise the epitope of interest
the secondary antibodies recognise the primary antibody
the region of primary antibody that is recognised by secondary antibody is constant among antibodies within a species, but different from species to species
what is immunoprecipitation?
recovery of protein complexes that bind to a specific antibody
the antibody:antigen complex is recovered by precipitation
the immunoprecipitate will contain the protein carrying the epitope recognised by the antibody AND any partner proteins stably associated with that protein
how does immunofluorescence microscopy work?
- prepare sample and place on microscopic slide
- incubate with primary antibody, wash away unbound antibody
- incubate with fluorochrome-conjugated secondary antibody, wash away unbound antibody
- mount specimen and observe in fluorescence microscope
inject purified mouse antibodies into a rabbit
the rabbit will recognise the mouse antibodies as foreign and make antibodies against the mouse antibodies
How does immunoblotting work?
antibodies are used to recognise individual protein species in a complex mixture of protein separated by SDS gel electrophoresis
1. the proteins are separated on the SDS polyacrylamide gel according to size
2. an electric current is passed through the gel, and the proteins migrate from the gel onto a membrane, and the SDS is washed away
3. antibodies are added to the membrane, they recognise the protein they are specific to (cannot be seen)
4. a secondary antibody is added, binds to the antibody, the proteins can now be seen (coupled to a fluorescent molecule or an enzyme that is colored)
called “sandwich” or indirect immunodetection: primary antibody is sandwiched between the secondary antibody and the protein
indirect detection of the protein
how does immunoprecipitation and co-immunoprecipitation work?
antibody specific to the protein is added to the mixture
a protein that binds to antibodies (attached to a bead) is added
by centrifugation, the precipitate is separated
the precipitate contains the complex of the bead/protein, the antibody and the protein complex to be isolated
what is green fluorescent protein (GFP) and how can it be used to identify proteins?
using GFP, we can analyse proteins in living cells
jellyfish have this protein that fluoresces green (its function is unknown)
this protein is an enzyme that can modify its own side chain, to make it look green
genes encoding GFP can be introduced into cells of any organism, (after the promoter region) and those cells will be producing GFP
