Exercise 4.2 - Separation of Protein Standards: SDS-PAGE
Figure 4.6 Protein standards and SDS-PAGE
Figure 4.7 Rf vs log molecular weight (Laemmli)
- 10% SDS-polyacrylamide gel from Exercise 4.1
- Protein standards 10
- 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 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
off the water.
- 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
- Add the same volume used in step 4 of SDS-sample buffer
or bromophenol 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
- 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
- Turn on the power supply and run the gel at 20 mA
constant current per 1.5 mm gel.
For example, if two gels are run, each having 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 one
of the spacers out from between the two glass plates. Using
the spacer or a plastic wedge as a lever, gently pry the
glass plates apart without damaging the gel contained
- 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 (Exercise 4.3 ), silver staining (Exercise 4.4),
enzyme detection (Exercise 4.7), Western blots (Exercise 4.9) or for more advanced procedur
es, such as
electroblotting or electroelution.
If prestained protein standards were used, the gels may
be scanned directly for analysis (Exercise 4.5 or Exercise 4.6).
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
If the gel is to be dried, use a commercial gel dryer,
such as the Hoefer SE540 or SE 1160 Slab Gel Dryer. Follow
the manufactuer's directions. Figure 4.6 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 weight.
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.
Figure 4.7 presents a plot of the relative molecular weight
of protein standards against the log of their molecular
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Cell Biology Laboratory Manual
Dr. William H. Heidcamp, Biology Department, Gustavus Adolphus College,
St. Peter, MN 56082 -- firstname.lastname@example.org