lab questions for finals

Question 1

why do proteins of the same size move through SDS at the same rate

Answer 1

native structure is unfolded by sds, making the shapes the same. the charge is also the same because they bind the same amount of SDS

Question 2

what moves faster in sds

Answer 2

the small proteins

Question 3

What does chromatin immunoprecipitation do

Answer 3

You find the cis-regulatory sequences occupied by a given transcription regulator. It uses an antibody against transcription regulator DNA sequences to precipitate transcription regulator protein bound sequences, remove the protein, then find where the sequence is.

Question 4

what does crosslinking do

Answer 4

covalent stabilization of protein-dna complexes

Question 5

what do you do after crosslinking in chip

Answer 5

membrane lysis. needs to get nuclear membrane too

Question 6

what happens after membrane lysis in chip

Answer 6

chromatin preparation, involving cutting it up into small pieces. can use sonication or enzymatic digestion

Question 7

how does immunoprecipitation get the protein of interest and dna bound to it out in chip

Answer 7

it forms an antibody-protein-dna complex, which is affinity purified using a resin that binds to the antibody.

Question 8

in chip, how do you get the protein off the dna

Answer 8

proteinase k, heat incubations, spin columns

Question 9

how do you get the final protein-dna levels in chip

Answer 9

using q-pcr

Question 10

in the gel shift (EMSA) assay, what moves slower, DNA or dna-protein complexes

Answer 10

DNA-protein complex. they beeeg. however, if the DNA is circular, the DNA-protein might actually go faster

Question 11

How do you label the DNA bands in EMSA assays

Answer 11

traditionally, radiolabelling was used. now, fluorescent probes and haptens

Question 12

what is a nonspecific competitor

Answer 12

an irrelevant, unlabelled nucleic acid used to prevent nonspecific proteins from binding to the target DNA

Question 13

what are binding reaction components

Answer 13

factors that increase the binding interactions between DNA and target protein. could be temperature, other conditions, or a molecule.

Question 14

what is a supershift

Answer 14

in gel shift assays, when a protein binds to dna, and that protein-dna complex binds to antibodies, making the complex even slower.

Question 15

what does a protein stain in western blotting do

Answer 15

it checks if the protein has moved out of the gel

Question 16

why do you need to wash the gel in western blotting

Answer 16

to remove background noise and unbound reagents

Question 17

what is used to detect the proteins in western blotting

Answer 17

traditionally used radiolabelling, now use chromogenic, fluorogenic, and chemiluminescent substrates

Question 18

what is the gene control region

Answer 18

because cis-regulatory sequences in eukaryotes are spread out so much, the gene control region describes the whole expanse for regulating and initiating transcription of a eukaryotic gene. includes promoter

Question 19

how do activator proteins increase the rate of transcription initiation

Answer 19

by attracting RNA polymerase II, and using DNA looping to put general transcription factors on activators. modify the chromatin to make it more accessible

Question 20

why do you need to release rna polymerases from the dna with activators

Answer 20

because sometimes they pause after 50 nucleotides or so and just stay there

Question 21

what happens if two transcription factors work together to facilitate transcription

Answer 21

synergistic effects. multiply rates of acceleration

Question 22

why does the condensate of transcription proteins stay loosely bound to DNA

Answer 22

because it’s very dynamic and things need to come and go

Question 23

what is a pglo plasmid, what parts does it have

Answer 23

araC (encoding for protein AraC), bla (resistant to ampicillin), GFP for fluorescence. origin of replication

Question 24

pglo system when there is no arabinose

Answer 24

ara c is not activated, will not bind to aral. rnap will not bind effectively either, very little transcription

Question 25

why is there less transcription of the pglo system when there is glucose present

Answer 25

because glucose reduces cAMP synthesis and CAPi synthesis, meaning less transcription. you need cap bound along with arac(a) for efficient rnap binding

Question 26

how does catabolite repression prevent e.coli from using other fuel when glucose is present

Answer 26

when there is glucose, there is low cAMP-CAP, so other pathways that don’t use glucose are not expressed

Question 27

most stable form of RNA

Answer 27

rRNA

Question 28

rate of migration for electrophoresis depends on

Answer 28

molecular structure, concentration of agarose

Question 29

DNA and RNA travel towards what in electrophoresis

Answer 29

travel towards positive electrode

Question 30

how can the percentage of agarose in agarose gels be changed for different DNA fragment size

Answer 30

higher percentage of agarose can be used for smaller fragments, vice versa

Question 31

what goes faster in electrophoresis assuming same charge

Answer 31

small molecules

Question 32

reporter gene

Answer 32

gene that researchers attach to a regulatory sequence of another gene of interest, aka GFP used in lab

Question 33

chelators, like edta bind what? what do they turn off as a result

Answer 33

mg2+, turning off rnases that require it

Question 34

lysozymes degrade what

Answer 34

cell wall

Question 35

potassium acetate and sds do what

Answer 35

precipitate protein and cell debris

Question 36

high salt disrupts

Answer 36

ionic conditions

Question 37

how are r-rnas made

Answer 37

by first transcribing them into a pre-rRNA which is then cleaved to form end products.

Question 38

what causes cytoplasmic streaming

Answer 38

movement of fluid due to movement of actin and myosin

Question 39

causes for browninan motion

Answer 39

random and irregular

Question 40

how do FDA and PI indicate which cells live and which are dead

Answer 40

fda in a living cell wil be converted into a green fluorescent molecule, fluorescein. but pi cannot go through living cell membranes so it does not color living cells

Question 41

features of FP-fused protein that should be verified before checking results of fluorescent studies

Answer 41

use monomeric fluorescent proteins (mFPs_ because oligermization might fuck with function. ensure no aggregation. ensure photostability, ensure FP is bright enough, does not change size or binding properties

Question 42

frap

Answer 42

if molecules move, bleached part will recover

Question 43

flip

Answer 43

photobleach continuously then see loss of fluorescence from the not photobleached part

Question 44

photoactivation

Answer 44

nonfluorescent or weakly fluorescent in native state, turns fluorescent when given a specific wavelength

Question 45

why is photoactivation better than photobleaching

Answer 45

faster and less intense light exposure, lower phototoxicity, better at imagining fast moving proteins. partial activation of fluorescence.

Question 46

palm

Answer 46

photoactivatable fluorophores inactive initially, laser activates them, then switched off. until entire sample is imaged.

Question 47

sted microscopy

Answer 47

excite, then bleach fluorescence around periphery. only small area fluorescing, finer details visible. allow study of organelles like mitochondria and synaptic vesicles in neurons.

Question 48

drosophila oogenesis

Answer 48

stem cell divides four times with incomplete cytokinesis, 1 of 16 become oocyte, others become nurse.

Question 49

purpose of nurse cells

Answer 49

mRNAs synthesized and then delivered. delivered assymetrically for differention of ovum that can complete development.

Question 50

pole cells in drosophila

Answer 50

formed around the posterior end of the syncytial blastoderm.

Question 51

how do you tell if the posterior cytoplasm is responsible for making germ cells

Answer 51

you transplant it and see if the germline determinants change the transplanted cell