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Lecture 16

BIOL 5060 Lecture Notes - Lecture 16: Methylation, Agarose Gel Electrophoresis, Deoxyribonuclease I

7 pages91 viewsSpring 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)
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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)
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