- 10% SDS-polyacrylamide gel
- Protein standards
- 2X-SDS sample buffer
- 1X-SDS electrophoresis running buffer (Tris-Glycine + SDS)
- 0.001% (w/v) bromophenol blue
- Micropipettes with flat tips for electrophoresis wells
- Remove the Teflon combs from the prepared gels by gently lifting the
combs from the chamber. Rinse the wells (formed by the removal of the
combs) with distilled water and drain it off.
- Fill the wells and the chamber with running buffer.
- Prepare aliquots of a known protein standard by mixing equal parts of the
protein standard with 2X sample buffer.
- Using a micropipette, add the sample to the bottom of a well. Add the blue
to a separate well.
- Remove the gel from its casting stand and assemble it into the appropriate
slab unit for running the electrophoresis. Be sure to follow the manufacturer’s
directions for assembly.
- Pour a sufficient quantity of running buffer into both the lower and upper
chambers of the electrophoresis apparatus until the bottom of the gel is
immersed in buffer, and the top is covered, while the electrodes reach into
the buffer of the upper chamber. Be careful not to disturb the samples in
the wells when adding buffer to the upper chamber.
- Assemble the top of the electrophoresis apparatus and connect the system
to an appropriate power source. Be sure that the cathode (+) is connected
to the upper buffer chamber.
- Turn on the power supply and run the gel at 20 mA constant current per
1.5 mm of gel.
For example, if 2 gels are run, each with 1.5-mm spacers, the current
should be adjusted to 40 mA. One gel with 1.5-mm spacers should be run
at 20 mA, while a gel with 0.75-mm spacers should be run at 10 mA.
- When the tracking dye reaches the separating gel layer, increase the current
to 30 mA per 1.5-mm gel.
- Continue applying the current until the tracking dye reaches the bottom of
the separating gel layer (approximately 4 hours).
- Turn off and disconnect the power supply. Disassemble the gel apparatus
and remove the glass sandwich containing the gel. Place the sandwich flat
on paper towels and carefully remove the clamps from the sandwich.
- Working on one side of the sandwich, carefully slide 1 of the spacers out
from between the 2 glass plates. Using the spacer or a plastic wedge as
a lever, gently pry the glass plates apart without damaging the gel
- Lift the bottom glass plate with the gel and transfer the gel to an appropriate
container filled with buffer, stain, or preservative.
The gel may at this point be used for Coomasie Blue staining, silver
staining, enzyme detection, Western blots, or more advanced procedures,
such as electroblotting or electroelution.
If prestained protein standards were used, the gels may be scanned directly
for analysis. Place the gel into 50% methanol and gently rock the container
for about 30 minutes prior to scanning. This can be accomplished by
placing the gels into a flat dish and gently lifting the edge of the disk once
every 30 seconds. There are commercially available rocker units for this
purpose. If the gel is to be dried, use a commercial gel dryer such as
(SE 1160 Slab Gel Dryer). Following the manufacturer’s directions
demonstrates a dried and stained gel containing a series of proteins of
known molecular weights.
- Plot the relative mobility of each protein against the log of its molecular
Relative mobility is the term used for the ratio of the distance the protein
has moved from its point of origin (the beginning of the separating gel)
relative to the distance the tracking dye has moved (the gel front). The ratio
is abbreviated as Rf. Molecular weight is expressed in daltons, and presents
a plot of the relative molecular weight of protein standards against the log
of their molecular weight.