2 ways to perform an agglutination test
- unknown cells with specific antibody
- known cells with unknown serum
diffusion assay
A ring of wells is cut into an agar plate, antibodies and antigen diffuse through the agar and will form visible precipitate between the wells. antibodies/serum or protein sample goes in the center
intersecting precipitin
non-identity
spur (wavelength looking thing)
partial identity
arc
identity
Describe immunofluorescent staining
- cells placed on slides
- cells fixed using paraformaldehyde or mathanol/acetone and rinsed
- cells permeabilized using dilute detergent
- cells blocked to reduce nonspecific binding
- primary antibody added and washed
- secondary fluorescent antibody added and washed
standard fluorescent microscopy
whole image is illuminated and out-of-focus light makes the in-focus image fuzzy; cheap but fuzzy and photodamage
confocal fluorescent microscopy
whole image is illuminated and out-of-focus light is blocked; good resolution but photodamage
two-photon fluorescent microscopy
less energetic light is used (infrared) and 2 photons must hit the dye simultaneously to excite it; lower photodamage and better tissue penetration but costly and inferior resolution to confocal
Are circular or linear chromosomes more complex?
linear due to end-replication problems
plasmids
small rings of DNA
