lab questions for finals Flashcards
why do proteins of the same size move through SDS at the same rate
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
what moves faster in sds
the small proteins
What does chromatin immunoprecipitation do
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.
what does crosslinking do
covalent stabilization of protein-dna complexes
what do you do after crosslinking in chip
membrane lysis. needs to get nuclear membrane too
what happens after membrane lysis in chip
chromatin preparation, involving cutting it up into small pieces. can use sonication or enzymatic digestion
how does immunoprecipitation get the protein of interest and dna bound to it out in chip
it forms an antibody-protein-dna complex, which is affinity purified using a resin that binds to the antibody.
in chip, how do you get the protein off the dna
proteinase k, heat incubations, spin columns
how do you get the final protein-dna levels in chip
using q-pcr
in the gel shift (EMSA) assay, what moves slower, DNA or dna-protein complexes
DNA-protein complex. they beeeg. however, if the DNA is circular, the DNA-protein might actually go faster
How do you label the DNA bands in EMSA assays
traditionally, radiolabelling was used. now, fluorescent probes and haptens
what is a nonspecific competitor
an irrelevant, unlabelled nucleic acid used to prevent nonspecific proteins from binding to the target DNA
what are binding reaction components
factors that increase the binding interactions between DNA and target protein. could be temperature, other conditions, or a molecule.
what is a supershift
in gel shift assays, when a protein binds to dna, and that protein-dna complex binds to antibodies, making the complex even slower.
what does a protein stain in western blotting do
it checks if the protein has moved out of the gel
why do you need to wash the gel in western blotting
to remove background noise and unbound reagents
what is used to detect the proteins in western blotting
traditionally used radiolabelling, now use chromogenic, fluorogenic, and chemiluminescent substrates
what is the gene control region
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
how do activator proteins increase the rate of transcription initiation
by attracting RNA polymerase II, and using DNA looping to put general transcription factors on activators. modify the chromatin to make it more accessible
why do you need to release rna polymerases from the dna with activators
because sometimes they pause after 50 nucleotides or so and just stay there
what happens if two transcription factors work together to facilitate transcription
synergistic effects. multiply rates of acceleration
why does the condensate of transcription proteins stay loosely bound to DNA
because it’s very dynamic and things need to come and go
what is a pglo plasmid, what parts does it have
araC (encoding for protein AraC), bla (resistant to ampicillin), GFP for fluorescence. origin of replication
pglo system when there is no arabinose
ara c is not activated, will not bind to aral. rnap will not bind effectively either, very little transcription
