L13 Flashcards
Techniques for identification
western blot, immunofluorescence
techniques for separation
immunoaffinity, immunoprecipitation
polyclonal antibodies
mixed pup of dif antibody molecs that recognize dif epitopes on the antigen
monoclonal antibodies
uniform pop of antibody molec that recognize the same epitope on the antigen
protein puritifcation
monoclonal antibodies against protein of interest
adsorption
highly speciifc
in favourable cases can be a one-step purification
elution
tight binding
can be drastic
often via pH change
western blot analysis
antibodies are usually labelled with an enzyme or a luminescent molec - rather than using radiolabelled antibody
primary and secondary antibodies for western blot analysis
primary - i.e. rabbit antibody against human protein
secondary - i.e. mouse antibody against Fc of rabbit one
immunoflourscence
observing in live cells
antibodies labelled with fluorescent gel`
in immunofluorescence how are actin filaments visualized?
antibody labelled with red fluorescent protein (RFP)
in immunofluorescence how are microtubules visualized?
different antibody to actin filaments - labelled with green fluorescent protein (GFP)
in immunofluorescence how are nuclei visualized?
blue fluorescent dye (DAPI) that binds DNA
immunogold labelling of thin sections
antibody is labelled with a gold colloidal particle and then visualized in electron microscope
catalysis requires _________
a high degree of atomic alignment
