1' Identifying Proteins in Crude Extracts by Western Blotting

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1. Identifying Proteins in Crude Extracts by
Western Blotting
2. Protein Bioinformatics
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Things To Learn From This Weeks Lab
THE FINAL PART OF THE PROTEIN WORK
The Western blotting Technique.
Antibodies are used to identify specific
proteins on Western blots, and specific
components of cells.
Bioinformatics-definition -analyses of protein
sequence using database -prediction of
subcellular localization -prediction of protein
function -prediction of domain structure and
function
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Electroblotting

• Voltage-driven transfer of proteins from a gel to
  a transfer membrane

Transfer Membrane
SDS Gel
Gel after blotting
Transfer Membrane After Blotting
Electro- blotting

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Western Blotting Detection of a Specific
Protein Using Protein-Specific Antibodies

• Also called an immunoblot.

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Antibodies are Proteins that are produced by an
animal immune system

• Each antibody recognizes and binds to one
  specific target molecule.
• The target molecule is called an antigen.

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Antibodies are Y-Shaped Proteins. Each antibody
has two binding sites for Antigens
Constant region is identical for all antibodies
made in the same species of organism.
Variable region (antigen binding site) is
different for different antibodies.
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Each Antibody can Recognize and Bind to a Single
Type of Protein (antigen) in a Crude Extract of
Thousands of Proteins.
proteins
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Antibodies Bind to Epitopes on an Antigen
Antibody
Epitopes on a protein are usually just a few
amino acids in size
epitope
epitope
Protein Antigen
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Polyclonal Antibodies Are Raised in animals such
as Rabbits or Goats for Research Purposes.

• The animal is injected with a purified protein.
• Over time, the animal develops a high titer of
  antibodies that recognize and bind to the
  injected protein.
• Blood is removed from the animal and the serum is
  used as the antibody solution.

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Polyclonal Antibodies
Monoclonal Antibodies
Inject Mice
Inject Rabbits
Antibodies that can recognize and bind to foreign
proteins are made. One hybridoma is selected for
production of an antibody that recognizes only
one epitope on the protein.
Blood serum fills with antibodies that can
recognize and bind to multiple epitopes on
foreign proteins
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Making Monoclonal Antibodies
Inject a potential antigen into a laboratory
mouse and allow the mouse to grow for several
weeks
Collect blood from the mouse and purify the
protein fraction that contains antibodies.
Purify the fraction of antibodies that bind to
the antigen that was injected.
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MONOCLONAL ANTIBODIES
Inject mouse with protein of interest
Fuse myelomas with lymphocytes
Myeloma Tumor Cells from Mouse Bone Cancer
immortal
Mouse spleen lymphocytes produce antibodies
against the protein
Mouse myeloma cells
Mouse lymphocytes
HYBRIDOMA
ANTIBODIES
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Direct Immunoblot

• A detectable label is attached directly to the
  antibody

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DIRECT IMMUNOBLOT
Protein X
Nitrocellulose membrane
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INDIRECT IMMUNOBLOT
Primary Antibody
Binds to Protein X
Secondary Antibody
Binds to the Primary Antibody
Is bound to the Secondary Antibody
Detectable Label
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INDIRECT IMMUNOBLOT

Goat Anti-Mouse Secondary Antibody
Label is conjugated to the secondary antibody
PRIMARY ANTIBODY
The labelled secondary antibody binds to the
constant region of the primary antibody
ANTIGEN
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INDIRECT IMMUNOBLOT PROCEDURE FOR DETECTING A
SPECIFIC PROTEIN ON A WESTERN BLOT
1. Block
2. Add Primary Antibody
3. Add Secondary Antibody
4. Detect the Label
Protein X
Nitrocellulose membrane
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WESTERN BLOT THE DETECTABLE LABEL IS AN ENZYME
THAT CATALYZES A REACTION THAT CAUSES A
BLUE-COLORED COMPOUND TO BE DEPOSITED AT THE SITE
WHERE ANTIBODIES ARE BOUND TO ANTIGENS.
NBT
Pi
P
Secondary Antibody
NBT
Primary Antibody
PROTEIN x
NBT Nitrobluetetrazolium
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Identification of Desmin Protein in a Crude
Extract of Chicken Gizzard Cells
Example of a Western Blot
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A control, without the primary antibody would
show

• A) the absence of the target protein.
• B) the non-specific binding of the primary
  antibody in the test sample.
• C) the failure of the secondary to recognise the
  primary.
• D) the non-specific binding of the secondary
  antibody.

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Antibodies can Also Be Used for In Situ
Observations of Cell Components
1. Raise an antibody against a specific cell
component
Example tubulin, or other proteins of the cells
cytoskeleton
2. Make the cell permeable and send the
antibodies in.
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For In Situ Observations, instead of using a
color reaction (as in the Western Blot) the
Detectable Label is a Fluorescent Molecule.
fluorescent tag
goat anti-mouse secondary antibody
fluorescent-labeled secondary antibody, ready to
sell to customers
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Once the Primary Antibody has entered the cells,
the excess is washed out and fluorescently tagged
Secondary Antibodies are sent in.
They find and bind to the primary antibodies.
Binds Primary Antibody
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The Cell is Viewed With a Fluorescence Microscope
GreenTubulin
A Human Cheek Cell Labeled with an Anti-Tubulin
Primary Antibody Which is Detected by a Goat
Anti-Mouse Secondary Antibody Conjugated to a
Fluorescent Tag.
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A Human Cell Tagged With Several Different
Fluorescently-Labeled Antibodies, Each
Fluorescing a Different Color
GreenTubulin
RedActin
Yellow Vimentin
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Summary

• Antibodies are proteins made by animals in
  response to antigen
• Can be polyclonal or monoclonal
• Antibodies are used in a laboratory to detect
  expression levels of various proteins
• Antibodies can be used in whole tissues/organisms
  to detect expression level of a particular
  protein
• Powerful tools for research

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Bioinformatics
The analysis of biological information using
computers and statistical techniques the science
of developing and utilizing computer databases
and algorithms to accelerate and enhance
biological research.
NIH Definition Research, development, or
application of computational tools andapproaches
for expanding the use of biological, medical,
behavioral or health data,including those to
acquire, store, organize, archive, analyze, or
visualize such data.
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Sequence databases

• Preserve
• Make available
• Inter-relate information

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Primary Sequence Databases
http//www.ncbi.nlm.nih.gov
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What can you do with all these sequences?

• Align/compare sequences
• Predict 10, 20, 30, 3D structures
• Predict domain(s) present in the sequence
• Predict subcellular location
• Predict post-translational modifications

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Aligning sequences

• Infer the function of a new gene by showing that
  it is similar to a previously characterized gene.
• This is called aligning the sequence of the
  encoded proteins or of the genes themselves.
• The function of even small parts of a protein
  (domains) can be inferred by alignment.
• Genome databases have made this a very powerful
  tool.

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Similarities can also be used to identify
functionally important regions.

• Active site of an enzyme.
• Ligand binding site of a receptor.
• DNA binding domain.
• Etc. etc. etc.
• Why should this work?
• Functionally important regions tend to be
  conserved. Changes may compromise the capacity of
  the protein to function.
• Danger!!!!!!!!
• In very closely related genes, conserved areas do
  not help identify function.

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BLASTBasic Local Alignment Search Tool

• One of the fastest methods of searching an entire
  database for regions of similarities.
• Good at finding domain similarities. Therefore it
  is good at classifying a gene based on function.
• Not a good tool for determining the best end to
  end (global) alignment of 2 sequences.
• Not a good tool to use for evolutionary
  comparisons.

Reference David W. Mount. 2001. Bioinformatics.
Sequence Genome Analysis. CSH Press.
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Different flavors of Blast

• Megablast - for nucleotides, long, highly
  similar. Use to find nearly perfect matches.
• PSI-Blast - Use it for finding distantly related
  relatives. For proteins. Search finds matches.
  Matches are aligned and produce an alignment
  model (HMM, template). Then search again. Keep
  going until no new entries are added.

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Different websites

• http//www.ncbi.nlm.nih.gov/
• http//us.expasy.org/tools/
• http//psort.ims.u-tokyo.ac.jp/form.html
• http//www.ebi.ac.uk/Tools/clustalw/index.html

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Summary

• Bioinformatics is a collection of powerful tools
  to analyze large amounts data sets
• Provides tools to investigate individual gene
  function in greater detail
• Also provides tools to gather and analyze data
  sets and formulate hypothesis for future research