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