Cell Biology Research Techniques Flashcards
Immunocytochemistry (ICC) and immunohistochemistry (IHC)
Specific antibodies against antigen A are coupled to fluorescent dye, gold particles, or another special tag to label them
Labeled antibodies bind to antigen and can be seen by microscopy
Enzyme-linked immunosorbent assay (ELISA)
Use antibodies to detect whole cells, proteins, or other antibodies in a sample
ELISA direct immunosorbent assay
Antibody is absorbed onto well and sensitizes plate
Test antigen is added; if complementary, antigen binds to antibody
Enzyme-linked antibody specific for test antigen binds to antigen, forming a double antibody sandwich
Enzyme’s substrate is added, and reaction produces a visible color change that is measured spectrophotometrically
ELISA indirect immunosorbent assay
Antigen is absorbed onto the well and sensitizes the plate
Test antiserum is added; if antibody is complementary, it binds to the antigen
Enzyme-linked anti-gamma globulin (anti-antibody) binds to bound antibody
Enzyme’s substrate is added: reaction produces visible color change that can be measured spectrophotometrically
Flow cytometry
Cells with certain antigens are labelled with fluorescent-antibody markers
Cells are suspended in a stream of liquid
Laser beam strikes each droplet
Fluorescence detector identifies fluorescent cells
Electrode gives positive charge to identified cells
Cells drop between electrically charged plates: cells with positive charge move towards negative plate
Separated cells fall into different collection tubes
Also called fluorescence-activated cell sorting (FACS)
Immunopurification
Purify protein of interest Connect antibody to bead Incubate bead slurry with cell homogenate (extract) Protein is in extract Ab binds to protein Beads are put into column Wash away everything not bound to bead (flow) Elute antigen (protein)
Western blotting
Antigen A is separated from other molecules by electrophoresis
Incubation with labeled antibodies that bind to antigen A allow the position of the antigen to be determined
Sensitivity increased by using multiple layers of antibodies
Sonication
Use high frequency sound to break open cells
Detergent
Dissolve membrane with chemical detergent: cells open
Homogenizer
Force disrupts membrane
Cells are sheared between a close-fitting rotating plunger and the thick walls of a glass vessel
Organelles are left intact
Pressure cell
Also known as French press
Force cells through small hole: disrupt membrane
Centrifugation
Separate homogenate into parts (fractions)
Homogenate is put into test tubes and rotated at high speed
Centrifugal force separates components based on size and density
Supernatant: smaller and less dense components
Pellet: larger and more dense components
Sedimentation coefficient
How fast a particle separates out of suspension
Size, surface area, density of molecule or organelle
Measured in Svedberg units (S)
Differential centrifugation
Spin supernatants at progressively higher speeds
Low-speed: pellet contains whole cells, nuclei, cytoskeletons
Medium-speed: pellet contains mitochondria, lysosomes, peroxisomes
High-speed: pellet contains microsomes and other small vesicles
Very high-speed: pellet contains ribosomes, viruses, large macromolecules
Velocity sedimentation
Components separate at different rates in sucrose
Test tube is filled with sucrose gradient (highest sucrose concentration at bottom)
Centrifugation: fast-sedimenting components are at bottom
Centrifuge tube is pierced at base: fractions are collected
