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Lecture 16

BIOL 5060 Lecture 16: 3:21_RecombinantDNATech_Hoffman

7 Pages
90 Views
Spring 2017

Department
Biology
Course Code
BIOL 5060
Professor
Hoffman
Lecture
16

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3/21_RecombinantDNATechnology_Hoffman3/21/2017 2:03:00 PM
The surface of a protein has the antigenicity that will raise
antibodies
= how proteins are detected by antibodies
o we can raise antibodies to an intact protein- whether it is a)
the native protein (wild-type, un-altered, normal protein) or
b) the tagged protein
o depending on the nature of the antibody, there are 3 levels of
quality
1) polyclonal sera- lowest quality antibody; B cells
produce antibodies to antigen; can produce 5 different
types of antibodies by 5 different types of B cells
Conserved regions and hypervariable regions
(with specificity)
Called polyclonal because there are different B
cells producing different antibodies- each B cell
only produces one of the antibodies
Issues: 1. the mouse may have had antibodies
before being injected with the protein; 2.
Antibodies to the protein of interest may cross
react with other proteins
Methods:
2) Affinity purification- bind the protein of interest to a
column, pass polyclonal sera over column, wash away
antibodies that cannot bind, elute affinity purified
antibodies
to get rid of the antibodies that weren’t raised to
your proteins
BUT doesn’t address cross-reactive antibodies
3) Monoclonal Antibodies- most pure reagent; take
mouse and isolate B cells, immortalize them with a vital
infection so they can grow in culture indefinitely and
separate them to generate clonal populations
monoclonal antibodies that screen for a single clone
we can screen for ones that detect our protein and not
others (WESTERN BLOT- only our protein’s bands will
light up)
find more resources at oneclass.com
find more resources at oneclass.com
Epitope tags- can add a site recognized by a
monoclonal antibody
HA- Hemaglutinin
Myc
V5- viral protein epitope
FLAG-
6 his- not great as an epitope but good for
purification
OR we can design a peptide based on the sequence of the protein
synthetic peptide
o Look for surface regions
Pros and Cons of working with a Native protein versus a Tagged
Protein
Your protein
Pros
Cons
Native
-Wild-type form
-Normal regulation of
production and normal
level of expression
-Looking at the protein in
its natural environment
- poor or no reagents for detection
-too little protein
Tagged
-There may be a
monoclonal antibody
available for detection
- Can be the only way to
have sufficient protein for
detection
-Altered expression may effect
outcome of expression- may lead
to over expression of your protein
-tag could alter function of protein
-could change where the protein is
located in the cell by changing the
proteins it interacts with
-could change folding, binding
partners, and location
Protein-DNA Interactions
Transcription or DNA replication-
Cloned a gene and want to study transcription of it
o Is there a protein or protein complex that binds upstream of
transcriptional start site? use EMSA
EMSA= Electrophoretic mobility shift assay (band-shift)
find more resources at oneclass.com
find more resources at oneclass.com

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Description
3/21_RecombinantDNATechnology_Hoffman 3/21/2017 2:03:00 PM The surface of a protein has the antigenicity that will raise antibodies • = how proteins are detected by antibodies o we can raise antibodies to an intact protein- whether it is a) the native protein (wild-type, un-altered, normal protein) or b) the tagged protein o depending on the nature of the antibody, there are 3 levels of quality ▪ 1) polyclonal sera- lowest quality antibody; B cells produce antibodies to antigen; can produce 5 different types of antibodies by 5 different types of B cells  Conserved regions and hypervariable regions (with specificity)  Called polyclonal because there are different B cells producing different antibodies- each B cell only produces one of the antibodies  Issues: 1. the mouse may have had antibodies before being injected with the protein; 2. Antibodies to the protein of interest may cross react with other proteins  Methods: ▪ 2) Affinity purification- bind the protein of interest to a column, pass polyclonal sera over column, wash away antibodies that cannot bind, elute affinity purified antibodies  to get rid of the antibodies that weren’t raised to your proteins  BUT doesn’t address cross-reactive antibodies ▪ 3) Monoclonal Antibodies- most pure reagent; take mouse and isolate B cells, immortalize them with a vital infection so they can grow in culture indefinitely and separate them to generate clonal populations monoclonal antibodies that screen for a single clone we can screen for ones that detect our protein and not others (WESTERN BLOT- only our protein’s bands will light up)  Epitope tags- can add a site recognized by a monoclonal antibody • HA- Hemaglutinin • Myc • V5- viral protein epitope • FLAG- • 6 his- not great as an epitope but good for purification • OR we can design a peptide based on the sequence of the protein synthetic peptide o Look for surface regions Pros and Cons of working with a Native protein versus a Tagged Protein Your protein Pros Cons Native -Wild-type form - poor or no reagents for detection -Normal regulation of -too little protein production and normal level of expression -Looking at the protein in its natural environment Tagged -There may be a -Altered expression may effect monoclonal antibody outcome of expression- may lead available for detection to over expression of your protein - Can be the only way to -tag could alter function of protein have sufficient protein for -could change where the protein is detection located in the cell by changing the proteins it interacts with -could change folding, binding partners, and location Protein-DNA Interactions Transcription or DNA replication- • Cloned a gene and want to study transcription of it o Is there a protein or protein complex that binds upstream of transcriptional start site?  use EMSA ▪ EMSA= Electrophoretic mobility shift assay (band-shift) ▪ You carry out a binding reaction with a labeled probe and protein or protein extract, run it on a gel that preserves an interaction ▪ Make a series of PCR products that overlap to walk us across the sequence (200 bp in length- if they’re too long it will bind different proteins and complexes; each product is of uniform length) ▪ Look at mobility to protein extract- they are labeled! This is only detected labeled DNA  Add cold probe to half of the sample before running it on the gel (other half will be solely protein extract with increasing concentration)  If we add equal amount of cold probe and protein, the band will be lighter by 50%/ will appear half as intense  If we add twice as much, it will be a third as intense  If we add 3 times as much, it will be 20% of original intensity  Proteins aren’t washed out until we put 50x as much DNA probe  If we added unrelated DNA it shouldn’t alter the interaction/ intensity at all  50x cold
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