Molecular Biology

Page and Western Blots



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Polyacrylamide Gel Electrophoresis, henceforth abbreviated PAGE, is an essential
tool in protein separation, esp. for a technique known as Western Blot, which
will be discussed shortly. Similar to agarose gel electrophoresis, the basis of
protein separation in PAGE is size - larger proteins move through a gel more
slowly than do lower molecular weight proteins. This rule applies generally,
but remember that proteins which are heavily phosphorylated or glycosylated will
move differently than unmodified proteins of the same molecular weight.
PAGE is the first step in a Western Blot, a technique in which the ultimate goal is the identification of a specific protein band within a sample containing a
multitude of proteins, e.g. a whole cell lysate. The gel set up in PAGE,
however, is more elaborate than in agarose gel electrophoresis. First the
separating gel is cast. This gel consists of water, bis-acrylamide, Tris buffer (to control pH), sodium dodecyl sulfate (SDS)(which swamps the native charge on each protein), ammonium persulfate (APS), and the polymerizing reagent
TEMED. The percentage of acrylamide in these gels ranges from 8-15% of the
final gel concentration. Lower percentage gels are more fragile but allow for a
faster separation time; higher percentage gels are more sturdy and often provide
better band resolution.
Casting the separating gel. Briefly, each reagent is added except for
TEMED. Once the gel apparatus is set up vertically and tested for leaks, TEMED
can be added to the mixture, which is then mixed and pipetted between the two
glass plates. A few minutes after pipetting the gel mixture, a layer of water
or butane should be carefully added on top of the gel to prevent drying.
Polymerization takes approximately 30 minutes.
Preparing the stacking gel. This gel will be added on top of the separating
gel and contain the wells into which the protein sample will be loaded. The
stacking gel contains the same reagents as the separating gel but with a lower
percentage acrylamide. Before pouring the stacking gel, ensure that the
separating gel has hardened and pour off the top layer of water/butane. Pipette
the stacking gel mixture quickly, as TEMED will cause rapid polymerization.
Insert a comb (most contain ten wells between the glass plates and wait 10-15
minutes.
The protein samples used in Western Blot are generally mixed with Lemmle
buffer (which contains the blue dye bromophenol blue) and a denaturing agent
such as DTT (dithiothreitol) or BME (beta-mercaptoethanol). Samples are boiled
for 3-5 minutes prior to loading. Molecular weight markers containing
multicolored standards are added to the extreme left or right lanes on the gel.
Most gels are run at 70-150V in a running buffer that consists of SDS, Tris, and
glycine. Please consult a protocol guide for detailed running buffer recipes.
After one to two hours, PAGE is complete and the gel is ready to be
transferred to a blotting membrane. The most commonly used material is PVDF
(polyvinyldifluoride); other membranes still in use include nylon and
nitrocellulose. The idea is to transfer proteins from the gel to the membrane
while preserving the exact separation pattern obtained in the PAGE step.
Transfer involves a transfer buffer containing Tris, glycine, and methanol.
Transfer cells work well at 100V for 60 minutes or 25-30V overnight, preferably
refrigerated.
The Western blot itself is relatively simple. Once the transfer cell is
disassembled and proper transfer of the proteins from the gel to the blotting
membrane has been confirmed, the blot is dipped in methanol and allowed to dry.
Blots can be stored for weeks to months at -20C if desired. To complete the
Western Blot, the membrane is blocked in a solution of 5% non-fat dry milk in a solution of Tris Buffered Saline (TBS) and the detergent Tween for at least 60 minutes. Next comes an incubation with the primary antibody for at least 60 minutes (or overnight with shaking in a refrigerator).
After three washes with TBS-tween, the secondary antibody is applied. This
antibody recognizes the primary antibody and contains a detection mechanism such
as a fluorescent tag, the enzyme HRP (horseradish peroxidase), or a radioisotope
such as iodine 125. After three more washes, the blot can be developed and
visualized with X-ray film in a dark room.
After the development step, the blot may either be discarded or stripped and
reprobed several times for other proteins.
Best of luck and happy blotting.

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