Flashcards
Exam 1 Prep
Cards in this deck (45)
- 1What is the Shine-Dalgarno Sequence?AGGAGGU
- 2What're 3 ways to determine protein conc.Lowry (Cu2+ is reduced causing color complex)
Nanodrop (280 nm)
Bradford (requires standard) - 3What is the Hayflick number?Number of times a cell will typically divide (40-60)
- 4What direction is DNA transcibed?3' - 5'
- 5What is the nuclear localization seq?PPKKKRKV
- 6Polyarginine would be a sequence used for?Penetrating the cellular membrane
- 7What're the steps for quantifying mRNA?Exploit the poly A tail
Use a poly T column to separate
rRNA and tRNA will separate with high salt
mRNA elutes with low salt
Quantify with Northern or rtPCR - 8How does a Northern Blot work?Isolate mRNA with elecotrophoresis
Probe with complementary DNA strand (P32 label) - 9Problems with mRNA levels?Translation is tightly regulated
mRNA easily degrades
can code for multiple proteins
doesn't account for PTM
doesn't address activity - 10How would you determine global RNA levels?Transciptional profiling
Arrays: physically match known and unknown oligonucleotides - 11C. elegans as model organismfull genome
defined fate for every cell
simple nervous systems and aging process - 12D. melanogaster as a model organismsmall and easy to grow
short generations
can lay up to 500 eggs
75 % of known human diseases genes have a match - 13Zebrafish as a model organismsimple vertabrate
transparent body
fins regenerate - 14African clawed frog as model orgvery large oocytes
can inject mRNA into oocyte
useful for electrophsyiological measurements
dis: complex genome, can't be bred over generations - 15Mouse as model orgfull genome, lab strains are identical, don't reject transplants
dis: long generation interval - 16Indirect large scale disease model generation vs direct large scaleuse x-ray or chemicals to generate mutations and then screen for phenotypes
requires osme prior knowledge about the cause of disease - 17Forward geneticsfind genetic basis of observed phenotype
- 18How do you go about reverse and forward genetics?Forward: random mutation, screen mutatns for novel phenotype, identify genotype that gives pehnotype
Reverse: mutation or deletion of a specific gene in genome, phenotypic analysis of deletion - 19How would you go about a mouse knockoutConstruct vector that contains gene of interest
insert into ES cells and create transgenic mouse
In addition to gene of interest use two resistance genes in order to select for the proper insertion. A resistance to determine if no insertion and another for proper insertion - 20What is the RNA intereference mechanism?RNA molecules inhibit gene expression be neutralizing targeted mRNA
dsRNA is cleaved by DICER in 21-23 frag w/ overhang
siRNA strans are separated and incorporated into RISC then bound to mRNA complement
mRNA is degraded by nuclease - 21Why use small molecules? in reference to chemical genetics?effect is reversible
allow temporal control
rapid effect
does response is typically achieved - 22[Cellular Imagine] Antibody imaging?Tough to produce antibodies, use one that recognizes epitope tag
- 23[Cellular Imagine] Localizing organic dyeHoechst dyes - bind in groove of DNA, locks into planar formation
EtBR - same - 24[Cellular Imagine] Fluorescent Protein?Fuse GFP to POI - large size of gfp may hinder function
Brainbow - exploits differing expression tot rack cells
RNA mimics - spinach - 25[Cellular Imagine] Organic dye that recognizes artificial protein tagFLAsh
-binds to CCXXCC
can be used in live cells
can label proteins
SNAP Tag
common 182 polypeptide
Halo-tag
fusion approach
smaller than SNAP - 26Analyte detection for probe design1) selectivity for target
2) turn on emission
3) biocompatible
4) fast kinetics - 27Fluorophore selection for probe designhigh optical brightness
visible or NIR excitation and emission
photostable
water soluble
high cellular retention - 282 strategies for imaging probeschelation based
-reversible Ca2+ sensor
Rxn based
irreversible
adv. signal build up - 29[Biomolecular interactions] Spectroscopic methodsFluorophore on ligan
fluorophore on amcromolecule
fluorescence polarization - rapid rotation of free -> loss of polarization during binding
binding of ligan to macro -> slower tumble ; less polarization - 30[Biomolecular interactions] Surface Plasmon ResonanceImmobilized ligan on surface
can measure dissocation from drop in signal
also binding speed - 31[Biomolecular interactions] Isothermal Titration CalorimetryHeat evolved or basord when two molecules interact
measure power needed to maintain reference cell at same temp as sample cell - 32[Biomolecular interactions] Mass Specnoncovalent complexes can be detected using ESI
DIS: can't determine Ka or Kd
false neg change - 33[Biomolecular interactions] NMRStrucutre activity relationships by NMR
ligands for subsite identify by NMR analogues screened to optimize binding
screen for ligan optimize ligan screen second ligand, optimize ligand link ligands
DIS: inattention to issues of dynamics, protein in soln isn't a protein in a cell - 34Photoaffinity labelingcovalent linkage is created between macromolecule and light-sensitive
[Pilot study technique]
need to confirm via mutagenesis - 35Characteristics of a good photoprobeSynthetic accessibility
stability to ambient light
useful activation wavelength (<300 nm)
photochemical excited stated with useful half-life
known photochemistry with useful products
reactivity with c-h and x-h bonds - 36Bioorthogonal rxn compoundsPhosphines ( PH3) , azides
- 37Native chemical ligation of proteins occurs between which reactive moietiesc-terminal thioester with n-terminal cys
[INSERT IMAGE] - 38What're the adv of solid phase synthesis?reagents can be added excess
byproducts and unused reagents can be washed away
no purification - 39How would you remove the solid support in SPSAcid labile -or photolabile (not great efficiency)
- 40What're Lipinski's 5 rules for drug design?Poor absorption or permeation more likely when:
5 H bond donors
10 H bond acceptors
Mw > 500
Compd is too nonpolar ClogP >5 - 41What're the 3 steps to HTS?Assay design, development, validation
Screening
Validation of hits - test in other assay - 42How does a time resolved FRET assay workOvertime, background from acceptor is lost and only donor fluorescence is present
[ADD IMAGE] - 43What're the adv of small molecule microarayrequires small volume of protein
stable for > 2 months - 44What is PAINSPan-assay interference Compounds
- filters out freq hitters that likely are false positives
-> filters are not widely accepted
-> aggregators are widely accepts - 45Cell based HTSAdv: compd in native environment
allows for differentiation/polymerization/complex phenotypes
dis: immortalized cell lines are different
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- Flash Cards based upon my initial Exam 1 Prep guide
- Created by
- Max Holle
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