Cell Molec Exam 2 Bagga

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phase contrast microscopy

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takes advantage of the refraction of light as it passes through the sample. It amplifies the path differences for greater resolution.

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differential interference contrast (DIC) microscopy

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bounces light off the surface of the sample

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Confocal scanning microscopy

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laser sections to build 3d model of sample

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TEM stain

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gold conjugated to antibodies

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Prep for SEM

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plating on mika, coat with evaporated carbon and platinum.

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Cell Sorter prep

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proteases eat connective proteins, EDTA chelates the ions that hold the cells together. Cells are mixed w/ antibodies conjugated to a fluorescent dye. (can also separate chromosomes)

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other cell sorting means

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adhesion to glass or irradiated plastic, or an antibody surface.

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primary culture, secondary culture

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taken from tissue, taken from primary culture

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passage

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each cell culture you make is a passage, and each lasts for 2-3 generations.

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"HeLa" cells

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transformed and immortalized human cells (host died in the 60s). important because her cells are the exception to mammalian surface cell growth (can grow in solution)

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COSMDS

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synthetic plasmid made from a lambda bacteriophage. COSMDS can be used in libraries for 40kb (or less) sections. Has one Ori.

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YAC

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yeast artificial chromosome. same idea as COSMDS but has multiple Ori, a telomere which can be separated in mitosis, it can handle much more info than COSMDS and slightly more than BAC because yeast are naturally more complex organisms.

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BAC

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bacterial artificial chromosomes. One ori. Can handle up to ~300kb.

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How would you test for:
lysosomes?
peroxisomes?
mitochondria?

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proteases
catalyases
dehydrogenases

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elute particles after ion exchange

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replace them with salts.

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"in silico"

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done on a computer chip

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what are the drawbacks of gene chips and microarrays?

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they are not built to distinguish between isoforms of the same gene product due to alternative splicing. Further, mRNA abundance does not necessarily correlate to protein expression abundance.

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SNP chips

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already being used to predict if chemotherapy will be needed after cancer surgery.

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Ultracentrifugation
Ultra-differential centrifugation
velocity & equilibrium sedimentation

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high speed
successive fractionations at different speeds
use of a density gradient

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how are cDNA libraries made?

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from mRNA and reverse transcriptase.

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partition chromatography

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seperates small molecules, uses differential solubility on an absorbent material, is analytical

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gel filtration chromatography

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column. preperative. separates by size - small molecules get stuck and move slowly. maintains bio activity.

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ion exchange

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cation exchange - cations are stuck in a negative matrix.
anion - anions are stuck in a positive matrix

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SDS PAGE

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essentially a "modified for proteins" version of DNA gel electrophoresis. SDS denatures proteins into long strands.

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two dimensional electrophoresis

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isoelectric focusing followed by sds page

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NMR

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disturbes magnetic structure to get an "image". Limited by size 15-20 kD only.

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Southrn Blot

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restriction cut your DNA, then run it on electrophoresis. transfer the DNA to nitrocellulose, probe with radiolabled ssDNA/RNA, perform autoradiography. (Finds DNA with radio-labeled ssDNA/RNA)

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Northern Blot

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extract RNAs, then run them on electrophoresis. transfer the RNAs to nitrocellulose, probe with radiolabled ssDNA/RNA, perform autoradiography. (Finds RNA with radio-labeled ssDNA/RNA) Same as southrn but for RNA

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Western Blot

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Immunoblot. Perform SDS PAGE with mix of proteins, and transfer to nitrocellulose gel. use primary antibodies to bind protein of interest, and secondary antibodies to bind and color the primary antibodies.

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south-western blot

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Southwestern because it finds DNA binding proteins. perform gel electrophoresis on proteins, transfer to nitrocellulose, probe with DNA to be bound. Maintains bio activity.

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north-western blot

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finds RNA binding proteins. mech is same as for south-western. biologically active.

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Homology - orthology, parology

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ortho - more closely related. alpha globin in humans and mice.
para - more divergent. alpha and beta globin in humans.

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BLAST

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for local alignments. useful for conserved regions of DNA

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Stains

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Ethidium Bromide = DNA
Coomassie Blue = proteins

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NF1

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regulates RAS, which is involved in the cell cycle.

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BRCA1

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breast cancer before 50 gene. its function was inferred from the cell cycle control gene RAD9 in yeast.