This page describes some theory behind the technique, a table
of useful standard proteins, procedures for electrophoresis,
a staining procedure for proteins. These are followed
by suggestions for data analysis, cleaning
up and equipment and reagents lists. You can also
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procedures in an appendix, the biochemistry lab
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Analysis of major biological phenomena at the level of macromolecules
often involves isolation and purification of a particular one from among
hundreds or thousands, assaying specifically for this molecule, determining
size (molecular weight), shape, and chemical structure. (Is it a polymer?
Is it electrically charged (ionic), neutral, polar or nonpolar?, etc.)
Several techniques have been developed which have made major contributions
to this type of study. Electrophoresis is used to identify and study
charged molecules and macromolecules.
One property of proteins and nucleic acids is that they are electrically
charged. Five of the 20 amino acids that commonly form proteins have either
acidic or basic side chains. These are ionized in water solutions at normal
pH, and therefore carry negative or positive electrical charges, respectively.
Since each type of protein has a unique sequence of amino acids, it probably
will also have a unique, net electrical charge in any set of conditions.
This fact is used to separate proteins and other macromolecules in cell
extracts, by the techniques called electrophoresis.
Samples of the material to be analyzed or purified are applied to a
supporting medium which may or may not aid in the separation. When subjected
to an electrical potential, charged particles migrate toward the electrical
pole opposite in charge to the net charge they carry, and at a rate that
is directly proportional to the magnitude of the charge they carry.
Thus different proteins migrate at different rates and will eventually
separate from others that may be present. This procedure can be used to
purify macromolecules and obtain material for further work, but is primarily
used to analyze and identify components.
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Standard and Unknown Samples
Standard proteins will be available in the laboratory, and are identified
by name, known molecular weight, pI and the net charge each carries at
the pH of the electrophoresis buffer we are using.
at pH 8.8
Bovine Serum Albumin (BSA)
Cytochrome C (CyC)
Obtain a sample of each known protein in a separate, labeled
cup in a clean spot plate (one drop from a Pasteur pipette is enough
for about 100 electrophoresis samples, so take only a small amount).
Always use separate pipettes or tips, etc. when sampling any solutions.
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Each group will be allowed one cellulose acetate strip on which to
apply eight (8) samples. Think about how to best use the 8 slots. For example,
what value might there be in applying mixed, known samples, or known mixed
with unknown samples, to a single slot (co-electrophoresis)?
Procedure: (for Gelman® Semimicro Electrophoresis Chamber)
- When you know which samples to apply where, pick up your slide with
the special forceps provided, and lower it (with the dull surface
up) slowly from one end into a tray of buffer until fully immersed.
- Allow to soak for 10 minutes or more. This is the most important
step in obtaining good results.
- While waiting, remove the Sepratek® applicator cover and fill the
rinsing well with deionized water (dH20).
- Place the applicator on the cover, but do not depress the button.
- Add 100 ml of cold buffer into each of the two chambers of the apparatus
(if not already filled).
- Just prior to application, use a clean WireTrole® capillary pipette
to apply an 8 µL sample of a single protein or mixture as desired
to the matted surface of each sample well in the applicator block. Spread
each sample to fill its well.
- Remove the strip and carefully blot between two pieces of filter paper.
- Transfer the membrane to the bridge, being careful that the two slots
near the end of the strip are centered around the two retaining keys at
the () end of the bridge.
- Quickly, install the bridge(s) in the chamber, matching the polarities
marked on both.
- Keep the chamber covered when not working within to keep the strips
Always Check To Be Sure The Power Supply Is NOT CONNECTED!
- Fit the applicator to the block and gently depress the button, hold
down for 5 seconds to completely fill the slots, and release the button
slowly to drain excess sample.
- Immediately fit the loaded applicator into the semi-micro bridge, and
apply the samples to the strip by fully depressing the button, holding
down for 5 s, and gently releasing it.
- Repeat to apply a second set of samples to the other side of your slide.
- Lightly mark the sample application points in pencil on the edges of
- Replace the chamber cover.
When all strips have been loaded, the instructor will connect the electrodes
to the power supply and begin electrophoresis at 100V for 20 minutes (or
at 150V for 15 min., if time is short).
Serum proteins are run for 45 minutes at 2 ma/strip and 300-350V (at no
time should current exceed 3 ma/strip or the voltage exceed 450V!).
- While electrophoresis is occurring, clean up the
applicator assembly. Rinse the applicator 5-10 times with clean dH2O
by gently depressing the button to lower the tips into the well on the
cover, and making a "water application" on absorbent paper. Be
extremely careful of the applicator tips as they are easily damaged. Do
NOT touch them with anything unless directed to do so by the instructor.
- Rinse the applicator block free of sample and blot dry with absorbent
- When electrophoresis is completed, turn off the power and disconnect
the cords BEFORE removing the chamber cover.
- Remove the strip, blot excess buffer from the ends, and look carefully
at the strip for colored protein bands.
- Mark the center of each with a very light pencil dot.
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Staining the strip
If quantitation of protein fractions is desired, follow the procedure
listed on p. 202 in Strong, F. & Koch, G. (1981) Biochemistry Laboratory
Manual, 3rd ed., Wm. C. Brown Co. Dubuque, Iowa.
- Immerse the strip in Ponceau S dye for 10 minutes.
- Rinse in 5% acetic acid for three separate rinses of at least 2 minutes
each with mild agitation.
- If necessary, strips may be stored in 5% HAc in the refrigerator.
- Strips may be preserved for a report by blotting, air drying and taping
to a report form with transparent tape.
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From the information given on the standard proteins and your results,
you should determine the identity of any unknown sample. Also determine
the net charge on all proteins, the distance migrated (in
millimeters) by each protein from the point of sample application,
and the magnitude of the charge on each protein relative to the
others from the pI - pH values. Since the pI is the pH value where the
net charge is 0, the larger the difference between pI and pH, the greater
the net charge on the protein, and the faster it should migrate in a given
electrical field. This assumes the media does not influence the migration,
as it does in polyacrylamide gel electrophoresis (PAGE).
Any report must contain your original strip or an accurately
duplicate (how?), showing the colored bands both before
and after staining. Be sure to consider the color
of any unknown samples, and the colored bands that may be visible before
staining. Why is this important?
You might consider the following points during your analysis:
- The key to determining what is present in an unknown sample
is to exclude those standards that can not be.
- How could you prove the identity of unknowns using one or more
slight variations of the techniques used in this lab? Think about co-electrophoresis.
- Discuss the importance of net and relative charges (pI
- pH values) on all proteins.
- Is there any correlation between the relative charges (pI - pH) and
the distance migrated for proteins? Explain.
- Is there any correlation between the molecular weight and the distance
migrated for the proteins? Explain.
- Explain any unusual or unexpected results. (HINT: Look at the Trypsin
- Present diagrams or slides carefully. Completely label the lanes, samples
applied, poles, application points and the pattern of bands before
and after staining:
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- 1 Gelman® Semimicro Electrophoresis Chamber with 3 bridges
- 1 regulated power supply
- Sepratek® 8 applicator assembly
- Super Sepraphore® strips
- - float strips, dull white face up, on cold electrophoresis buffer
(add 1 mL ethanol/100 mL buffer to improve wetting) until fully wet, then
immerse completely and store in refrigerator until used.
- White spot plate, pasteur pipettes and bulbs
- flat tipped forceps
- Absorbent pads (or filter paper) - 3" x 6"
- 10 µL Drummond Wiretrol® capillary pipettes
- 5 staining trays - 1 dye, 3 HAc (5%) in line in hoods, one with buffer
Solutions containing Trypsin should be made just prior to use and stored
frozen. Lysozyme precipitates on freezing, but seems to redissolve on thawing
if mixed thoroughly.
- 1-2 L 5% HAc - 50 mL glacial acetic acid into 950 ml dH2O.
- 1200 mL Gelman® Buffer, pH 8.8 - Dissolve 18 g (1 vial) of buffer
salts in 1200 mL dH2O and store in refrigerator for 1-2 weeks.
Reusable 4-5 times. Keep cold prior to and during use.
- 200 mL Ponceau S dye - 0.5% dye in 5% TCA - as supplied by Gelman or
add 0.5 g dye to 100 mL of 5% Trichloroacetic acid. Refrigerate for 1-2
- 10 mL each - 1% w/v protein solutions (10 mg/mL) - 0.l g protein and
10 mL sterile dH2O or 0.15 M sodium acetate (NaAc).
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Laboratory Clean Up:
The Sepratek® applicators should be carefully cleaned and inspected
prior to storage.
Do not leave the acid in the cover well any longer than necessary and
be very careful of the applicator tips. Do not immerse the applicator itself
in ANY solution.
- Add a few mL of 10% nitric acid to the cover well and push the applicator
button to soak the tips for a while.
- Apply the acid to filter paper to ensure all slots are clean.
- Replace the acid in the well with dH2O and push the applicator
button to soak the tips for a while.
- Rinse the applicators with 5-10 dH2O washes and blotting
- Air dry and store in cover, block assembly.
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Last Modified on 7/1/98