Chapter 4: Electrophoresis - Endnotes

1. We refer to molecular orientation. It is also possible to separate whole cells or organelles on the basis of their surface charge.

2. Also known as sodium lauryl sulfate or SLS.

3. 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.

4. 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.

5. 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.

6. These can be easily manufactured from 22 gauge needles with the points ground off. Never use pointed needles when working with acrylamide!

7. 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 air bubbles.

8. 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.

9. 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.

10. Prestained molecular weight standards are available from several sources; BRL, Rockville, Md, Bio-Rad or Sigma Chemical Co, St. Louis.

11. 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.

12. 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.

13. 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.

14. 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 visually.

15. Fotodyne Inc. New Berlin, Wisconsin.

16. 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.

17. Directions given for Isco Model 1312 Gel Scanner (Isco, Inc. 4700 Superior, Lincoln, NE 68504).

18. 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.

19. 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 this exercise.

20. C.L. Markert and F. Moller 1959.

21. 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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