Research Methods in Cell Biology

what kind of cell are bacteria?

prokaryotes

what kind of cells are yeasts?

eukaryotes

what cell line is 3T3?

fibroblast in mice

what cell line is HeLa?

epithelial cell in human (cervical cancer cells)

what cell line is 293?

kidney in human (transformed with adenovirus)

what cell line is CHO?

ovary in chinese hamster

what does growth of primary human cells look like?

grows fast in phase 1, remains constant in phase 2, decreases in phase 3

what does an immortalized mouse cell look like?

has a dip at around 30 days of initiation of culture

what separates different types of cells from each other?

fluorescence activated cell sorting

FACS

fluorescence activated cell sorting

what is done to cells in FACS to distinguish them?

stained with fluorescent dye

how many cells are in one drop in FACS?

one or less

what is an example of a cell separated by FACS?

T cells from white blood cells

will terminally differentiated cells divide more?

no

are embryonic cells differentiated or undifferentiated?

undifferentiated

what can embryonic stem cells be used for?

reproductive or therapeutic cloning

what is more useful reproductive or therapeutic cloning?

therapeutic cloning

what kind of tissue can totipotent ES cells make?

all tissues, including placental

what kind of tissue can pluripotent cells make?

all tissues except for placental

can pluripotent cells be used for reproductive cloning? why or why not?

no, because they cannot make placentas

what are hybridoma cell lines made for?

to make monoclonal antibodies

what is the purpose of using hybridoma cells?

to allow for a continuing source of anti-”X” antibody which would otherwise die in a culture by itself

what are monoclonal antibodies specific for?

one part of a molecule usually a protein

examples of monoclonal antibody usage

immunoaffinity chromatography, western blots, ELISAs, immunofluorescent staining, immunoprecipitations, immuno-gold labeling

how can small coated vesicles be purified?

binding of antibody for a vesicle protein and linkage to bacterial cells

what is used for visualizing cells?

microscopy

what are optical microscopes configures for?

bright-field, phase contrast, epifluoresence

how can live cells be visualized?

microscopy techniques that generate contrast by interference

what are the three methods of visualizing live cells?

bright field simple light, phase contrast in phase light canceling, nomarski/DIC polarized

what are double-label fluorescence microscopy used to visualize?

relative distribution of two proteins

deconvolution

out of focus fluorescence eliminated computationally

deconvolution fluorescence microscopy

yields high-resolution optical sections that can be reconstructed into one 3-D image

confocal microscopy

produces an in-focus optical section through thick cells

what does confocal microscopy eliminate?

light from out of focus planes

what can help explore the function of genes in cultured cells?

RNAi screens in combination with microscopy

electron microscopy

images are formed from electrons that pass through a specimen or are scattered from a metal-coated specimen

how does transmission electron microscopy show fine features?

by looking at negatively stained samples

what makes surface details or small objects visible through transmission electron microscopy?

metal shadowing

what are gold particles coated with protein A used for?

detecting antibody bound protein by transmission electron microscopy

what are some examples of organelles viewed by transmission electron microscopes?

rough ER, clathrin coated pit, golgi complex

what are some examples of organelles viewed by transmission electron microscopy of thin sections?

structures in mitochondria and structures in chloroplasts

scanning electron microscope

carbon coated sample has electron beams scattered at an angle detected to produce a 3D image

what must cells be to be viewed under SEM?

dead and dehydrated

separating sub-cellular components

differential centrifugation is a common first step in fractionating a cell homogenate

how can a mixed organelles fraction be further separated?

equilibrium density-gradient centrifugation

low speed centrifuge

sediment cells out of solution

high speed centrifuge

purify nuclei, sediment bacteria, precipitates

ultra centrifuge

separate organelles and molecules

what is true of ultra centrifuges?

they are spun so fast that a vacuum must be created

what centrifuge speed pulls out whole cells of solution?

low speed

how are nuclei and cytoskeleton isolated in centrifugation?

cells are lysed then spun at low speeds

how is the supernatant isolated

spun at high speeds to isolate other organelles

what does low speed contain?

whole cells, nuclei, cytoskeletons

what does medium speed contain?

mitochondria, lysosomes, peroxisomes

what does high speed contain?

microsomes, small vesicles

what does very high speed contain?

ribosomes, viruses, large macromolecules

how are sucrose gradients used with ultra centrifuges?

separating molecules of different densities

column chromatography

separates molecules into fractions with eluent

what are the three types of beads used in column chromatography?

ion exchange, gel filtration, affinity chromatography

what does ion exchange separate?

charged molecules

what does gel filtration separate?

molecules by size

what does affinity chromatography separate?

specific molecules to antibodies or other molecule specific substrates crosslinked to the beads

what molecule is able to pass through size exclusion?

large unretarded molecule

what type of chromatography is considered the best?

affinity

sodium dodecyl sulfate

ionic detergent which denatures and coats proteins giving them a negative charge

beta mercaptoethanol

reducing agent that breaks disulfide bonds

what chemical tools help stabilize proteins?

sodium dodecyl sulfate and beta-mercaptoethanol

how are individual polypeptide components of a protein separated?

by SDS polyacrylamide gel electrophoresis

what stain is used for SDS polyacrylamide gel electrophoresis?

coomasie

what occurs in 2D polyacrylamide gel?

isoelectronic focusing

what occurs after isoelectronic focusing?

turned 90 degrees and separated by size in SDS page

can 2D acrylamide gel electrophoresis be combined with western bloting?

yes

what can mass spectrometry be used for?

identify proteins

what do fusion proteins do?

used for localization, purification or to look at protein-protein interaction

how are fusion proteins created?

different tags

western blot

identify protein from a preparative gel based on spot position

what is used for the yeast two hybrid technique?

BAIT and PREY

what does the yeast two-hybrid technique do?

detect protein-protein interactions

what is x-ray crystallography used for?

determining protein structure

what is NMR spec used for?

determining protein structure

restriction enzymes

restriction enzymes cut DNA at specific sequences/sites

what can restriction enzyme cut DNA be ligated to make?

new recombinant molecules

gel electrophoresis

separates molecules by size

acrylamide use

separates small molecules and single base-pair size differences

agarose use

large molecules, pulse field gels for whole chromosomes

probe

a single-stranded sequence of DNA or RNA used to search for its complementary sequence in a sample genome

genomic DNA library

inserts

cDNA library

a collection of cloned DNA sequences that are complementary to the mRNA that was extracted from an organism or tissue

polymerase chain reaction

amplifying large amounts of small pieces of DNA quickly in a test tube

PCR

polymerase chain reaction

the sequence of at least the ends of the DNA piece must be…

known to make oligonucleotide primers

first cycle of PCR

producing two double-stranded DNA molecules

second cycle of PCR

producing four double-stranded DNA molecules

third cycle of PCR

producing eight double stranded-DNA molecules

DNA fingerprint

technique used to determine paternity or to find a region linked to a genetic trait

what does a low percent of ddNTPs result in?

occasional termination

how are terminated DNA fragments separated?

by size

how are genes found?

by looking for open reading frames

ORF

open reading frames

what is the reading direction for sequence of both bottom and top DNA strand?

N terminus to C terminus