Separate proteins by SDS-PAGE for Western blot


The first step in many protein
analytical methods, including Western blot
analysis, is to separate the proteins in a sample by gel
electrophoresis. The most widely used protein electrophoretic technique is sodium dodecyl sulfate
polyacrylamide gel electrophoresis, or SDS-PAGE, in which proteins are denatured
and evenly charged by the ionic
detergent SDS so that the proteins migrate through the gel based only on
size. Polyacrylamide gels can be
poured in the lab by combining various
concentrations of acrylamide and bisacrylamide
with the crosslinker ammonium persulfate
and TEMED, which catalyzes the
polymerization reaction. Pre-cast polyacrylamide gels are
also available with varying acrylamide
concentrations and gradients. Let’s look at performing
SDS-PAGE using HeLa cell lysates and
pre-cast gels. After adjusting protein levels
so that all lysates have a uniform
protein concentration, combine the lysates with a
sample buffer that includes SDS, glycerol, a
tracking dye such as bromophenol blue and a
reducing agent such as dithiothreitol,
beta-mercaptoethanol or TCEP. Heat the samples for 3-5 minutes to denature and reduce the
proteins and then cool them to room
temperature. Load a polyacrylamide gel into
an electrophoresis unit, and fill the upper and lower
buffer tanks of the unit with a running
buffer made of Tris, glycine and SDS to enable
proper flow of the electric current through
the gel. Then load the protein samples
into the wells in the gel. Because the glycerol in the
loading buffer is heavier than the running
buffer, the samples should easily drop to the bottom of their
respective wells. The volume loaded into each well
can range from 1uL to over 40uL, depending on
the protein concentration, well size
and gel thickness. Also, load a separate well with
a molecular weight marker to
assess the relative molecular weight of the target
protein. Once the samples and marker are
loaded, connect the electrophoresis unit
to a power source, making sure that the cables are
correctly connected to ensure correct protein
migration. Then apply an electric field at
the appropriate voltage or current, which
depends on the type and number of gels being used. Bubbles appearing around the
cathode indicate that the current is
flowing. Once the dye front reaches the
bottom of the gel, turn the power off and unplug
the cables from the power source. Remove the gel cassette from the
electrophoresis unit and carefully separate the gel
from the plates. The gel is now ready for Western
blot, Coomassie stain or another
protein analytical method.

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