Chapter 4: Electrophoresis - Endnotes
We refer to molecular orientation. It is also possible to
separate whole cells or organelles on the basis of their
Also known as sodium lauryl sulfate or SLS.
Guidelines given by John A. Smith, in Current
Protocols in Molecular Biology, Section II, 10.2.2
"Electrophoretic Separation of Proteins". Greene Publishing
Associates, Brooklyn 1987.
The term disc is applied to discontinuous gels, but
unfortunately has two meanings when applied to
electrophoresis. Some investigators continue to use an older
reference to the discoidal front found in tube gels, and
thus use disc interchangeably with the terms column or
tube. Modern usage of the term refers to the term being
applied to a discontinuous pH system which may be run in a
tube or slab configuration.
If a continuous pH gradient is employed, the
stacking gel is eliminated, the buffers are different, and
the technique is referred to as zone electrophoresis. If
the molecules being separated are allowed to migrate to a
particular zone due to a pH gradient in the gel, the
procedure becomes known as electrofocusing.
These can be easily manufactured from 22 gauge
needles with the points ground off. Never use pointed
needles when working with acrylamide!
Alternatively, add a stirring bar to the flask
and stir on a magnetic mixer at low speed. It is important
that the solution be stirred slowly so as to not introduce
If problems are encountered using water at this
step, water saturated isobutanol may be substituted. To aid
even further, a little Oil Red O dye can be added to the
isobutanol for visual distinction. Since the alcohol is less
dense than water, it layers more readily without mixing with
the gel. It must be thoroughly rinsed out before adding the
stacking gel, however.
Teflon combs come in a variety of configurations
for sample volume and number of lanes (3,5,10,15 or 20). Use
a comb suitable for the number of samples to be run.
Prestained molecular weight standards are
available from several sources; BRL, Rockville, Md, Bio-Rad
or Sigma Chemical Co, St. Louis.
The SDS/sample buffer contains glycerol and
consequently the sample will have a higher density than the
SDS/separating buffer. It is best to use a flat tipped
micropipette designed specifically for this application, but
any flat tipped syringe needle will also work. The volume of
sample will depend on the size of the wells, but typically
is 25-100 µl. Some experimentation will be
necessary to obtain the proper volume.
In some applications, the bromphenol blue is
added to the SDS/sample buffer and thus this step is
unnecessary. Bromphenol blue is known as "tracking" dye and
is used to monitor the progress of the electrophoresis
separation. A drop of 0.1% phenol red may be substituted.
Alternatively, prestained protein standards may be used
without a tracking dye, although this is not recommended.
10x10x2 glass baking dishes are useful for this
purpose, or ordinary refrigerator containers of appropriate
size. While the former will need a covering of plastic wrap,
the latter come with lids.
If tube gels are used, the apparatus used for
separation can be modified for stain removal if slightly
larger tubes can be fitted. Place a gel that has been washed
in water and 7% acetic acid (1 hr.) into a tube that has had
a 0.5 cm gel plug formed in the tip. Place the gel into the
tube with the origin up, that is away from the plug, and
fill the tube with 7% acetic acid. Insert the tube into the
appropriate apparatus and fill both upper and lower chambers
with 7% acetic acid. With the upper pole as the cathode,
apply a 15 milliamp current per tube. The stain will wash
into the lower bath and the progress can be monitored
Fotodyne Inc. New Berlin, Wisconsin.
Measurements can be made easier if the image of
the gel is first enlarged such that the distance from the
point of origin to the front is 10 cm on the final print.
This can be done with a photographic enlarger, and a print
may not be required.
Directions given for Isco Model 1312 Gel Scanner
(Isco, Inc. 4700 Superior, Lincoln, NE 68504).
Alternatively, a cruder yet effective method, is
to cut the peaks from the paper tracing and weigh the paper
for each peak. This must be done at one period of time to
prevent changes in the weight due to humidity. The technique
is surprisingly accurate, however. This process can also be
used with photographs of the gels, without the use of an
expensive gel scanner.
Human serum works well for this exercise, and students may
use their own blood. To avoid the obvious problems in
obtaining safe samples of human blood products, substitute
fresh sera from laboratory animals. A rabbit or rat will work,
but the LDH patterns will be different than those given in
C.L. Markert and F. Moller 1959.
From Enzyme-Linked Immunoelectrotransfer Blot
Technique (Western Blot) for Human T-Lymphotropic Virus Type
III/Lymphadenopathy-Associated Virus (HTLV-III/LAV)
Antibodies VC.W.Tsang, K.Hanclck, M. Wilson, D.F. Palmer,
S.D. Whaley, J.S. McDougal, S. Kennedy. Immunology Series
No. 15 Procedural Guide. U.S. Dept. of Health and Human
Services. December 1986.
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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