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Lecture 16

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

7 pages91 viewsSpring 2017

Course Code
BIOL 5060

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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
= 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
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
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
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
V5- viral protein epitope
6 his- not great as an epitope but good for
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
Your protein
-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
-There may be a
monoclonal antibody
available for detection
- Can be the only way to
have sufficient protein for
-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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