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The Popular Technology SDS PAGE & Western Blotting:: Principle and Application
The Popular Technology SDS PAGE & Western Blotting:: Principle and Application
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Western blot analysis can detect your protein of
interest from a mixture of a great number of
proteins (a cell can contain 30,000 different
proteins - and these same proteins can even be
altered giving you over 300,000 different proteins!).
an idiom in Chinese
(大海撈針)
Western blotting can give you information about
the size of your protein (with comparison to a size
marker or ladder in kDa), and also give you
information on protein expression (with comparison
to a control such as untreated sample or another cell
type or tissue).
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Western blot analysis can analyze any protein sample
whether from cells or tissues, but also can analyze
recombinant proteins synthesized in vitro.
Figure 1. Details of the steps involved in obtaining protein for western blot.
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Definition: SDS-PAGE is an abbreviation for sodium dodecyl
sulfate (SDS) polyacrylamide gel electrophoresis (PAGE)
Electrophoresis is a technique for separating,
SDS-PAGE
• Standsfor “SDS Polyacrylamide Gel Electrophoresis
• Similar in many ways to agarose gel electrophoresis
that we used to separate DNA molecules:
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SDS-PAGE
4) Smaller molecules move faster through the gel than
larger molecules
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In forming the polyacrylamide gel, acrylamide monomers polymerize into long chains that
are covalently linked by the crosslinker N, N'-methylene-bis-acrylamide (bis for short).
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Analysis of protein samples by SDS polyacrylamide-
gel electrophoresis and Western blotting
Protein
bands
detected by
specific
antibody
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To Detect your Protein:
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Table of Contents, in order done for western blot:
Steps in Western Blotting:
- Western Blot
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To Prepare Samples for Western Blot: continue
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Lysing:
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Denature Protein Samples :
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SDS-PAGE Gel Preparation for Western Blot
5–7 % 29–150 kD
8–10 % 14–66 kD
13–15 % <36 kD
18–20 % <20 kD
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Gel Electrophoresis
Load MW marker
Gel Electrophoresis
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How an SDS-PAGE gel is run
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Tranfer of Proteins to Membrane for Western
Blotting
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Assemble the immunoblot sandwich
(-)
sponge on black
?
PVDF membrane
filter paper
Gel
filter paper
sponge
(+)
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How Does Western Blotting Work?
· A Protein Sample
· A Good Antibody to Detect your Protein of Interest If your protein is a novel
protein, you must produce an antibody yourself or get a company to do it for you. In this case you
will need at least a small amount of your protein either purified from cell extracts or made as a
recombinant (ie in vitro or in a recombinant protein expression system).
Primary antibody
Wash
Secondary antibody
Wash
detection
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Diagram 1
Membrane (carrier)
Diagram 2
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Diagram 2 shows a western blot example gel.
As you can see the protein in lane 3 has a higher expression than
the normal sample in lane 5, which is interesting.
Also, the protein spots in lanes 3 and 5 are the same size as the
2nd spot in the size ladder from lane 1.
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If the membrane is placed into the water too quickly, air
will be trapped and will appear as white blotches in the
membrane. Protein will not transfer onto these areas.
Place into distilled water slowly, with one edge at a 45◦◦
angle. This wetting procedure works for nitrocellulose and
nylon membranes only.
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chromogenic
Chromogenic- substrate
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Chromogenic detection
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Table 10.8.3 Applications of Immunoblotting
Application Comment
Antibody development
and characterization Characterize antibody specificity
Subcellular
localization of an
expressed protein Analyze isolated organelles (e.g., Golgi apparatus, plasma
membrane, nucleus) for presence of specific proteins; probe
intact cells and tissue for subcellular localization.
Diagnostics Separate and blot a viral lysate to test serum for antibodies
that bind key viral proteins, indicating presence of antibodies
against an infection (e.g., HIV testing)
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Application Comment
Protein sequencing by
mass spectrometry Characterize the readily available proteins blotted onto
membranes using sensitive methods, e.g., peptide sequencing
by MALDI (Carr and Annan, 1996)
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Fuzzy Bands, Bands Smeared, Bands not Sharp
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Materials
0.2 M NaOH
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Flow chart of Western blotting
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