Chapter 8 Methods
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Cell Culture
isolating cells from tissues
primary cell lines
-cells taken from live tissues, organs, etc -limited lifespan
secondary cell lines
-cells taken from primary cell lines -finite # of divisions
immortalized cell lines
-cell lines altered to live forever and divide forever, bypass senescence
senescence
cell permanently stops dividing
Hybridoma Cell Lines
differential normal + mouse tumor cells are centrifuged with fusing agent - forms heterokaryon, media filters out and only allows heterokaryon to go through - gets cloned as hybrid cells, process for creating immortalized cell lines
Monoclonal
from single clone
Polyclonal
from multiple clones
Flow Cytometry
analyze individual cells in liquid suspension, measures physical and molecular properties
FACS
subset of flow cytometry that physically separates cells based off of fluorescent markers
Protein Purification
isolate specific protein from complex mixture
Organelle Isolation
separates different organelles
Sedimentation
different speeds from centrifuging will seperate different organelles through pellets
Density Centrifugation/ Fractionation
samples are layered and organelles match based on density buyoncy
Chromatography
used to seperate proteins by charge, size, specific bonding, hydrophobicity
Electrophoresis
separates proteins by using electrical field to move charged molecules
Western Blotting
uses antibodies to detect specific proteins
Mass Spectrometry
identifies and characterizes macromolecules by their mass to charge ratio
PCR
-tests if nucleic acid is present and how much -amplifies DNA sequence, creates millions of copies from DNA fragments
FISH - Fluourescent in Situ Hybridization
detect and locate specific RNA + DNA sequences within cells or tissues using Fluorescently labelled probes - exactly what’s inside nucleic acid
RT-PCR
quantifies RNA levels in samples by converting RNA to cDNA and amplifies -how much RNA is present
Microarrays
measures expression levels of thousands of mRNAs
RNA interference
silences or reduces expression of specific genes by targeting mRNA for destruction
transient vs stable
transient = temp, stable = permanent