Protein Electrophoresis

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 jump to other lab procedures in an appendix, the biochemistry lab schedule, my home page or jump to the addresses at the bottom.

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.
Proteins Suggested:
Molecular Wt.
Net Charge
at pH 8.8
alpha-amylase (Ama)
97,600 (48,200)
Bovine Serum Albumin (BSA)
Hemoglobin (Hb)
Cytochrome C (CyC)
Lysozyme (Lys)
Trypsin (Try)

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. Why?

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Sample Application:
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)
  1. 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.
  2. Allow to soak for 10 minutes or more. This is the most important step in obtaining good results.
  3. While waiting, remove the Sepratek® applicator cover and fill the rinsing well with deionized water (dH20).
  4. Place the applicator on the cover, but do not depress the button.
  5. Add 100 ml of cold buffer into each of the two chambers of the apparatus (if not already filled).
  6. 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.
  7. Remove the strip and carefully blot between two pieces of filter paper.
  8. 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.
  9. Quickly, install the bridge(s) in the chamber, matching the polarities marked on both.
  10. Keep the chamber covered when not working within to keep the strips from drying.
    Always Check To Be Sure The Power Supply Is NOT CONNECTED!
  11. 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.
  12. 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.
  13. Repeat to apply a second set of samples to the other side of your slide.
  14. Lightly mark the sample application points in pencil on the edges of the strip.
  15. 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!).
  16. 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.
  17. Rinse the applicator block free of sample and blot dry with absorbent paper.
  18. When electrophoresis is completed, turn off the power and disconnect the cords BEFORE removing the chamber cover.
  19. Remove the strip, blot excess buffer from the ends, and look carefully at the strip for colored protein bands.
  20. Mark the center of each with a very light pencil dot.

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Staining the strip

  1. Immerse the strip in Ponceau S dye for 10 minutes.
  2. Rinse in 5% acetic acid for three separate rinses of at least 2 minutes each with mild agitation.
    1. If necessary, strips may be stored in 5% HAc in the refrigerator.
  3. Strips may be preserved for a report by blotting, air drying and taping to a report form with transparent tape.
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.

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Data Analysis:

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:

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Equipment Needed:
Reagents Needed:
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.
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Laboratory Clean Up:

The Sepratek® applicators should be carefully cleaned and inspected prior to storage.

  1. Add a few mL of 10% nitric acid to the cover well and push the applicator button to soak the tips for a while.
  2. Apply the acid to filter paper to ensure all slots are clean.
  3. Replace the acid in the well with dH2O and push the applicator button to soak the tips for a while.
  4. Rinse the applicators with 5-10 dH2O washes and blotting applications.
  5. Air dry and store in cover, block assembly.
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.
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  Last Modified on 7/1/98

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