Re: How do Western blots work?

From Wei Wong:
The SDS-PAGE gel consists of two different sections: the stacker and the 
separator. Both sections are mixtures of acrylamide, bisacrylamide, TEMED, 
APS and Tris-HCl.  They both contain pores that are formed by acrylamide 
molecules linked together by bisacrylamide in a lattice-like structure.  If 
simply mixed together, acrylamide and bisacrylamide do link together in a 
process known as polymerization, but at a very slow rate.  Since free 
radicals accelerate the polymerization process, TEMED and ammonium 
persulfate are added since TEMED causes the production of free radicals 
from ammonium persulfate.  The amount of acrylamide and bisacrylamide in 
the gel determines how big the pores will be, which in turn affects how 
fast proteins move through the gel.  Thus, a large protein will move faster 
through a gel containing a small amount of acrylamide/bisacrylamide 
(because it has bigger pores) compared to one containing a high amount of 
acrylamide/bisacrylamide (because it has smaller pores).  Tris-HCl acts as 
a buffer, and is a source of chloride ions (the significance of this will 
become clear later on).  One of the main differences between the stacker 
and separator is that the stacker usually contains a low amount of 
acrylamide and bisacrylamide, while the amount of acrylamide and 
bisacrylamide in the separator is varied depending on the size of the 
protein you're interested in.  Also, the stacker has a lower pH (6.8) than 
the separator (8.8).

Although the gel is obviously crucial to SDS-PAGE, buffers also play an 
important role.  Before the proteins are loaded onto the gel, they are 
mixed with a buffer called the sample buffer whose key components are 
Tris-HCl, SDS and a reducing agent such as DTT or beta-mercaptoethanol and 
placed in boiling water.  This causes the protein to assume a linear 
structure, which allows it to bind SDS (larger proteins bind more SDS than 
smaller ones), become negatively charged and therefore move towards the 
cathode in the presence of an electric current.  The buffer that touches 
the top of the stacking gel and the bottom of the resolving gel and allows 
an electric current to be conducted through the gel is called the running 
buffer.  It is the source of glycine for the SDS-PAGE process.

When the proteins in sample buffer are loaded onto the gel and an electric 
current is applied, they get trapped in what is termed the moving boundary 
while migrating through the stacker.  Chloride ions from the Tris-HCl in 
the stacker and the sample buffer form the front part of this boundary, 
while glycine molecules from the running buffer form the back part of this 
boundary.  The proteins get sandwiched between the chloride ions and the 
glycine molecules and form a very thin zone, or stack.  When this moving 
boundary reaches the resolving portion of the gel, the difference in pH 
between the stacker and the separator causes the glycine molecules to 
ionize and the glycine ions move through the protein stack right behind the 
chloride ions.  Freed from the moving boundary, the proteins move through 
the separator, the distance covered being dictated by the size of the 
protein and the size of the pores in the separator.

Reference: Maniatis et al.  Molecular Cloning: A Laboratory Manual.  Cold 
Spring Harbor Laboratory, 1989.

Building off of Wei's very thorough description of SDS-PAGE, here's the rest
of the procedure for western blotting:

After the samples have been run through the polyacrylamide gel, the gel is 
removed from its apparatus, and placed in a transfer apparatus, which is 
very similar to the running apparatus, except the current is run from the front
to the back of the gel instead of top to bottom.  The gel is placed in the
transfer apparatus sandwiched between layers of filter paper and sponge (to hold 
it flat and upright), with a membrane placed directly against the gel on the
side facing the positive electrode.  This membrane is usually a nylon mesh
coated with either nitrocellulose of polyvinydene fluoride (PVDF) that binds
strongly to the proteins as they migrate out of the gel.  After a couple of 
hours of current, all of the protein should be off the gel and bound to the 
membrane.  The membrane is then removed from the transfer apparatus and rinsed
in buffered saline to remove anything non-proteinaceous that may be stuck to
it.  This membrane is then the blot - it contains all of the proteins in the
same bands in the same lanes and the original gel, but it a much more stable
form.  Now for the western part:

The western analysis involves detecting specific proteins on a protein blot
by using antibodies against the proteins.  Simply, the blot is placed in 
buffered saline containing an antibody against a known protein for some
period of time dictated by the quality of the antibody.  The membrane is then
rinsed to remove unbound antibody, and then placed in a new solution contain-
ing a secondary antibody.  Secondary antibodies are antibodies that specifically 
recognize other antibodies and are attached to a label, usually an enzyme.  
Thus, the secondary antibody can convert the first antibody's binding to the 
protein into a detectable signal through its label.  In the simplest scenario,
the secondary antibody is attached to an enzyme that changes the color of a
chemical.  In this case, the blot is rinsed to remove unbound secondary anti-
body, and then placed in a solution containing this chemical, which turns a
different color on the blot wherever the specific protein is present.

More interesting than the technique (I think) is the origin of the term "western" 
blot, which can be found in this previous post:
http://www.madsci.org/posts/archives/may97/864186493.Ge.r.html