Chemiluminescence
refers to a chemical reaction that results in the emission of light.
Electrophoresis
Electrophoresis
is a method used to separate molecules based on their size and charge.
In separating proteins, polyacrylamide gels are used. Since polyacrylamide
gels consist of a network of polymers that contain spaces (pores) between
them, the pores can act in a manner similar to a sieve.
The pore size
decreases with increasing gel monomer concentration. Large molecules tend
to migrate more slowly through the gel. When one performs SDS
polyacrylamide gel electrophoresis (SDS-PAGE), the process results
in the separation of proteins into discrete bands along the gel. SDS-PAGE
is the most common method used to separate proteins. The technique was
further developed by Laemmli in 1970. His paper, published in Nature,
is the most cited manuscript in the journal's history.
Horseradish
peroxidase (HRP)
HRP is an enzyme that is conjugated to secondary antibodies
and aids in the detection of the bound proteins.
Isoelectric
Focusing
Isoelectric focusing is another electrophoretic separation
method. Since the net charge of a protein is determined by the pH of its
environment, isoelectric focusing allows for the separation of proteins
in pH gradients. As the proteins move along the gel of varying pH, their
net charges change. When the net charge is zero, the proteins will stop
migrating. This point is known as the isoelectric point.
Membranes
The
two most common membrane types used for western blots are nitrocellulose
and polyvinylidene difluoride (PVDF). Nitrocellulose was one of the first
membrane types used for western blotting. Its advantages are that it is
easily wetted and it produces good immunoblotting results. PVDF membranes
were developed by Millipore Corporation in 1985. Compared with nitrocellulose,
PVDF membranes exhibit improved protein retention under harsh conditions
(i.e. in the presence of organic solvents or under acidic or basic conditions).
The greater mechanical strength of PVDF membranes is an asset when one is
interested in stripping and reprobing for further immunodetection.
Non
Fat Dry Milk (NFDM)
NFDM (often called blotto) consists of milk proteins
that are used as blocking agents in order to help reduce background signal
in immunoblotting. Blocking with milk tends to provide a better signal to
noise ratio.
Stripping
Buffer
Stripping
buffer is applied to break antibody-antigen interactions, allowing for reprobing
of the membrane without performing further SDS-PAGE. Already probed membranes
are gently agitated in the stripping solution, and immunoblotting is then
started - begining with the blocking (NFDM, blotto) step.
Tween-20
Tween-20 is a nonionic detergent that is used in washing
buffers and antibody solutions to help reduce background.
