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WESTERN BLOT
Reagents:
2x SDS buffer
Running buffer
Transfer buffer
Blocking buffer
1st and 2nd antibodies
What is a buffer?
• A buffer is an aqueous solution consisting of a mixture of a weak
acid and its conjugate base or a weak base and its conjugate acid.
• HA + XOH → HOH (water) + XA (salt)
• Weak acid →
conjugate base
•
Its pH changes very little when a small amount of strong acid or
base is added to it and thus it is used to prevent changes in the pH
of a solution. Buffer solutions are used as a means of keeping pH at
a nearly constant value in a wide variety of chemical applications.
Many life forms thrive only in a relatively small pH range so they
utilize a buffer solution to maintain a constant pH.
• One example of a buffer solution found in nature is blood.
Where are Proteins found in cells?
• Most proteins are found in the cytoplasm of the cell,
however, some can be found inside the nucleus.
• Proteins are large biological molecules consisting of one or
more chains of amino acids. Proteins perform a vast array
of functions within living organisms, including catalyzing
metabolic reactions, replicating DNA, responding to stimuli,
and transporting molecules from one location to another.
Proteins differ from one another primarily in their
sequence of amino acids, which is dictated by the
nucleotide sequence of their genes, and which usually
results in folding of the protein into a specific threedimensional structure that determines its activity.
Lysis Buffer
• Different Lysis buffers are used to either lyse the
cell membrane to release the protein in the
cytoplasm or to lyse both the cell membrane and
nuclear membrane to release proteins in the
cytoplasm and inside the nucleus.
• The Lysis buffers used to lyse the cell membrane
are gentle detergents, while those which lyse the
nuclear membrane are strong detergents.
Running Buffer
The running buffer provides a mobile path for
the electrical current that carries the samples
traveling through the gel.
It contains very little SDS which gently coats the
amino acid chains, allowing it to travel through
the gel with the continuous electrical current.
Transfer Buffer
• Transfer buffers must enable both effective
elution of proteins from the gel matrix and
binding of the protein to the membrane.
• The choice of transfer buffer depends on the
membrane being used and the physical
characteristics of the protein of interest
Blocking Buffer
• Traditional protein blocking buffer contains
BSA, casein and milk for Western blotting,
ELISA and other immunoassay detection
methods with micro plates and membranes.
• The protein blocking buffer is used to block off
nonspecific binding sites so that the
antibodies used is just specific to attach to the
particular protein of interest.
1st and 2nd Antibodies
• 1st antibody is a protein which is specific to attach to the
protein of interest from the cell.
• 2nd antibody is another protein which has a horse radish
peroxidase enzyme that reacts with hydrogen peroxide to
produce a chemiluminescence reaction that releases light,
which in turn captured by a charged couple camera.
• The 2nd antibody is specific to the 1st antibody and the
chemiluminescence reaction locates the location of the
protein of interest on the membrane after the Western blot
is run.
The Principles of Western blot
• The western blot (sometimes called the protein
immunoblot) is a widely accepted analytical technique used
to detect specific proteins in the given sample of tissue
homogenate or extract. It uses gel electrophoresis to
separate native proteins by 3-D structure or denatured
proteins by the length of the polypeptide. The proteins are
then transferred to a membrane (typically nitrocellulose or
PVDF), where they are stained with antibodies specific to
the target protein, which in turn give out light due to a
chemiluminescence reaction for the detection of the
location of that specific protein.
• The proteins are separated by size (in kD) compared to a
colored marker on the gel.