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Axiva Sichem Biotech - Laboratory Filtration Products
Axiva Sichem Biotech - Laboratory Filtration Products
SYRINGE FILTERS
Membrane Filters
Reveals Protein
of Interest
Western Blot
(Immunoblotting)
A technique for
detecting specific
proteins separated by
electrophoresis by use
of labeled antibodies.
So called Since it has
some similarity to a
southern blot.
Definition
The Western Blot is
an analytical
technique used to
detect specific
proteins in a given
sample of tissue
homogenate or
extract.
Advantages of Western
Blot
Western blot analysis
can analyze any protein
sample whether from
cells or tissues, but
also can analyze
recombinant proteins
synthesized in vitro.
Western blot is
dependent on the
quality of antibody you
use to probe for your
protein of interest, and
how specific it is for
this protein
Gel Electrophoresis
Uses gel electrophoresis to
separate native or denatured
proteins by the length of the
polypeptide (denaturing
conditions) or by the 3-D
structure of the protein (native /
non-denaturing conditions). The
proteins are then transferred to a
membrane (typically
nitrocellulose or PVDF), where
they are probed (detected) using
antibodies specific to the target
protein.
Monoclonal and Polyclonal
Antibodies are used
Tissue Preparation
Gel
Electrophoresis
Transfer
Blocking
Detection
Analysis
Samples may be taken from whole tissue or from cell culture.
In most cases, solid tissues are first broken down mechanically using a
blender.
Another method for transferring the proteins is called electro blotting and uses an
electric current to pull proteins from the gel onto the PVDF or nitrocellulose
membrane.
Blocking
The membrane has the ability to bind to proteins in this case both the target and
antibodies are proteins and so there could be some unwanted binding.
The protein in the dilute solution attaches to the membrane in all places where the
target proteins have not attached. Thus, when the antibody is added, there is no room on
the membrane for it to attach other than on the binding sites of the specific target
protein.
Detection
During the detection process, the
membrane is “Probed” for the
protein of interest with a modified
antibody which is linked to a
reporter enzyme, which when
exposed to an appropriate substrate
drives a colorimetric reaction and
produces a color.
WESTERN BLOT REACTIVITY IN ONE HIV- 1, SEROCONVERTER
Analysis
After the unbound probes are washed away, the western blot is ready
for detection of the probes, that are labeled and bound to the protein of
interest.
A western blot is also used as the definitive test for Bovine Spongiform encephalopathy
(BSE, commonly referred to as ‘mad cow disease’)
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