Exam 1 Part 3 Flashcards
parts of antibody
light chain variable region hinge heavy chain constant region
antigen
foreign particle (protein) that the Ab recognizes
Epitope
region/part of the antigen to which the antibody binds
1 antigen can have MANY epitopes
B cells
produce antibodies
each is unique
normal role of antibodies
recognize antigen and clump together so rest of immune system can get rid of it
make antibodies specific to protein of interest
- purify antigent
- find appropriate animal and inject
- animal immune system makes antibodies
- blood draw
polyclonal antibodies
many different epitopes recognized
monoclonal
single epitope recognized
made by combining with tumor cell to create hybrid cell
using antibodies to study cells/proteins
can look at protein expression, isolate protein, means of comparison, structure/function, or target cells in body
immunoprecipitation
when: to isolate your protein of interest
1. conjugate an antibody to a bead
2. add cell lysate to column
3 antibody binds to protein
4. wash column
5. elute off your protein (can change heat, pH, chemicals, etc)
Immunocytochemistry and immunohistochemistry
- label
- fixation/prep
- incubation, wash
- view
amplification of signal
primary antibody not strong enough signal, so secondary antibody that binds to primary constant site added
ELISA
Enzyme-linked immunosorbent assay
antibody sticks to stuff
ELISA direct
put Ab on plate add antigent add another Ab color "is antigent present?'
ELISA indirect
Ag
add sample
2o Ab added with marker
color reaction
flow cytometry
count cells that you have labeled
based on charge
Fluorescence-activated cell sorting
FACS. sort cells into tubes
Cell breakage
sonication
detergent
homogenizer
pressure cell
sonication
high frequency sound waves break open cell
detergent
chemical dissolves cell membrane (SDS, trident, etc)
Homogenizer
physical way to open cells
Pressure cell
force cells through small opening
french press
Centrifugation
separate homogenate into fractions
Sedimentation coeffienct
Measured in Svedberg units
Differential centrifugation
centrifuge at different speeds
velocity sedimentation
put homogenate into a solution of sucrose
equilibrium sedimentation
density gradient centrifugation
1 density of sucrose
