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Lecture 13

BIOL 5060 Lecture 13: 3:28_RecDNATech_Hoffman

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Department
Biology
Course Code
BIOL 5060
Professor
Hoffman

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3/28_RecombinantDNATechnology 3/28/2017 7:19:00 PM
What protein binds to this sequence?
We have knowledge of the cis element not the trans
One hybrid (budding yeast)
o There are 2 reporters
o This sequence has a His3 reporter
We want the new plasmid to be able to have GAD
protein bind
Not really interested in proteins function, just if it can
bind
Transform yeast to leu+ media library
Screen for reporter expression
Xgal blue colinies
Insert cDNA in leu+ media to make a library
o Very little His3 expression can produce a His+ colony (so we
have to make sure there aren’t false positives?)
3- aminotriazole His3 inhibitor
addition of 3AT to this medium requires higher level
of expression for growth to occur
When is my protein bound to the DNA in cells?
Want to make sure we analyze in vivo
Chromatin Immunoprecipitation (ChIP)
o Take cells and treat with formaldehyde to create covalent
bonds (cross link) between proteins and the DNA to which
they are bound
o Isoalte DNA
o Physically shear to ~500 bp
o Immune precipitate protein of interest (need Ab)
o Reverse crosslink (to free DNA from protein)
o Detect region of interest vs. control region of DNA look for
enrichment of region of interest
1) shear DNA IP there are some random sequences that make
is through a control is oligos around gene
o those that don’t make it through go through PCR
o Controls: oligos to a distant site, sheared chromatin without
IP
2) Endpoint PCR- a specific number of cycles
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o compare control region to region of interest ratio
enrichment of site of interest
o Pro- good visual
o Con- not good quantitatively
o PCR of semi. PCR is better for measurement
Protein-Protein Interactions
Do protein X and Y interact?
o Co-immunoprecipitation (coIP)
Make protein extract
Use Ab to bind protein X to precipitate it
Run immunoprecipitation on gel
Try to detect both Protein X and protein Y by Western
blot proteins are separated before running on gel
May also try reversing the AB IP protein Y, detect
protein C
o Use of Recombinant DNA
Problem: The AB could precipitate with protein (by
itself)
Epitope rags rather than Ab to native protein
Using promoters to over-express proteins to facilitate
detection
Use clones of these genes to allow purification
Generate Ab to native protein
o Pulldown vs. co-IP
Replace first IP w. affinity purification
6 His- cobalt column (Talon bends)
GST
MBP (maltose binding protein)
CBP (Chitin binding protein)
Two Hybrid screen
Q1- do X and Y interact?
Q2- Who interacts with W/X?
Have to make your own hybrids
o Each transformant has on hybrid protein
X protein will be used as bait
find more resources at oneclass.com
find more resources at oneclass.com

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Description
3/28_RecombinantDNATechnology 3/28/2017 7:19:00 PM What protein binds to this sequence? • We have knowledge of the cis element not the trans • One hybrid (budding yeast) o There are 2 reporters o This sequence has a His3 reporter ▪ We want the new plasmid to be able to have GAD protein bind ▪ Not really interested in proteins function, just if it can bind ▪ Transform yeast to leu+ media library ▪ Screen for reporter expression ▪ Xgal blue colinies ▪ Insert cDNA in leu+ media to make a library o Very little His3 expression can produce a His+ colony (so we have to make sure there aren’t false positives?) ▪ 3- aminotriazole His3 inhibitor ▪  addition of 3AT to this medium requires higher level of expression for growth to occur When is my protein bound to the DNA in cells? • Want to make sure we analyze in vivo • Chromatin Immunoprecipitation (ChIP) o Take cells and treat with formaldehyde to create covalent bonds (cross link) between proteins and the DNA to which they are bound o Isoalte DNA o Physically shear to ~500 bp o Immune precipitate protein of interest (need Ab) o Reverse crosslink (to free DNA from protein) o Detect region of interest vs. control region of DNA look for enrichment of region of interest • 1) shear DNA IP  there are some random sequences that make is through a control is oligos around gene o  those that don’t make it through go through PCR o Controls: oligos to a distant site, sheared chromatin without IP • 2) Endpoint PCR- a specific number of
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