Analyzing Cell, Molecules and Systems 1 Flashcards
What is a cell culture?
refers to the removal of cells from an organism, and promoting their subsequent growth in a favorable artificial environment
What are the types of cell culture?
primary cell culture
established or continuous cell culture
What is a primary cell culture?
derived directly from animal
involves enzymatic and/or mechanical disruption of the tissue and some selection steps to isolate the cells of interest form a heterogenous population
How long do primary cell cultures survive for?
finite period of time
What are some examples of primary cell cultures?
primary neurons, cardiomyocytes
What is an established or continuous cell line?
primary culture that has been made immortal by transformation, most commonly tumor derived or transformed with a virus
What are examples of a established cell line?
SH-SY-5Y (human neuroblastoma derived), CHO, Hela
What are the advantages of cell cultures?
- study of cell behavior without complexity/variation from live animals
- cell characteristics maintained over several generations
- control of growth environment leads to uniformity of sample
- cultures can be exposed to reagents or protective agents
What are the disadvantages of cell cultures?
- need to develop/ standardize techniques to maintain healthy cells
- quantity of material is limited
- dedifferentiation and selection may occur, altering original cellular mechanism/pathway
What are the applications of cell cultures?
- basic science research on cell/gene/protein function
- simulation of disease in vitro
- testing of drugs/vaccines/ chemicals
- chromosomal or genetic analysis
- production of biological products
- regenerative medicine
What is protein purification used for?
to study the unique structure and function of individual proteins
in subcellular fractionation, what happens to the tissues?
mechanical blending
What does the homogenate do in subcellular fractionation?
suspension of different cell types
What does centrifugation do in subcellular fractionation?
separate different cell types based on size and density
What does lysis of cells do in subcellular fractionation?
osmotic shock, ultrasonic vibration, mechanical blending, forcing through small orifice
What does ultracentrifugation do in subcellular fractionation?
separate organelles
What is column chromatography?
a column is packed, and a sample is added, followed by a solvent which separates the sample to get desired products
What are the three types of column chromatography?
ion-exchange
gel-filtration: size based
affinity: substates bind to protein
What are techniques used to analyze proteins?
SDS -PAGE
western blotting
ELISA
mass spectrometry
What is SDS
largely hydrophobic with negative charge
unfolds proteins and gives all proteins uniform charge
What does beta mercapatoethanol do?
reduces disulfides
What can SDS PAGE do?
visualize separated proteins with stains/dyes
What does western blotting do?
analyzing specific/known proteins using antigens
What does ELISA do?
tests for the levels of specific antigen or antibody concentrations in biological samples using a corresponding antibody or antigen
What is indirect ELISA?
measures the amount of antibody in a sample
What is sandwich ELISA?
measures the amount of an antigen in a sample
What does mass spectrometry do?
identify unknown proteins
What does the pellet contain after low speed centrifugation?
Whole cells
Nuclei
Cytoskeleton
What does the pellet contain after medium speed centrifugation?
Mitochondria
Lysosomes
Peroxisomes
What does the pellet contain after high speed centrifugation
Microsomes
Small vesicles.
What does the pellet contain after very high speed centrifugation?
Ribosomes
Viruses
Large macromolecules
