Troubleshooting for Gel Electrophoresis

Polyacrylamide gel electrophoresis, or PAGE, is a technique commonly used for separation of organic molecules in a biological sample. Molecules with lower size and weight migrate faster on a polyacrylamide gel when an electrical current is applied. Clear distinct bands are generally observed after the completed gel is incubated in a particular stain. It is a relatively simple method. However, absence of bands or bands that smear or are unclean could indicate a problem in one or more steps of the method. You should follow basic guidelines to troubleshoot a gel electrophoresis method when your results are not as expected.

Things You'll Need

• Quality grade bis: acrylamide or precast plates
• Two glass plates
• Acrylamide: Bis
• TBE Buffer (TRIS, Boric Acid, EDTA)
• Power supply
• Upper buffer chamber
• Lower buffer chamber
• Protein of DNA sample
• TEMED
• Ammonium persulfate
• Gel stain

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Instructions

• 1

  Start with the final step of gel electrophoresis first and work backward. For gel electrophoresis, the final step is the staining procedure used to observe sample bands. Few problems should actually occur during the staining procedure. However, powdered stains should be well dissolved in the appropriate buffer before they are used to stain a gel. Ascertain the correct concentration of stain was used.

• 2

  Check the power supply. The absence of bands could indicate the current or voltage setting was too high and the test samples migrated off the gel. High settings can also cause sample components to migrate through the gel too fast, causing poor resolution and bands that appear unclean. Samples may not migrate through the gel if the power supply setting is low. Samples can also drift from the well where they were loaded and not migrate through the gel.

• 3

  Check the liquid acrylamide used to cast the polyacrylamide gel. Typically, the percentage of acrylamide used to cast a polyacrylamide gel ranges from 5 to 15 percent. Samples comprised of larger molecules are loaded on a 5 percent gel because large fragments move slower on gels with a higher acrylamide percentage. Small molecules are loaded on a higher percent gel because a low gel concentration would not be capable of resolving smaller samples.

• 4

  Check the reagents used to polymerize the acrylamide. A polyacrylamide gel is formed by polymerization of a liquid mixture consisting of bisacrylamide and acrylamide. Ammonium persulfate and TEMED are added to the acrylamide to catalyze gel formation. Typically, the acrylamide should show signs of polymerization (gel formation) 15 minutes after ammonium persulfate and TEMED have been added. Polymerization that occurs too quickly is the result of high amounts of ammonium persulfate. Too little ammonium persulfate can cause incomplete or uneven polymerization.

• 5

  Confirm the quantity of the samples loaded. High concentrations of samples may move sluggishly through the gel and reduce band resolution. A sample concentration that is low may not be observed after the gel has been stained. These stains have limitations as to the minimum amount of sample that can be imaged as a result of stain sensitivity.

• 6

  Confirm quality of the samples loaded. Salts and other contaminants present in a sample can affect the movement of the sample along the gel. Electrophoresis is the result of electrical current running through a gel that contains a current-conducting buffer; high salt concentrations in the sample can affect the movement of this current through the gel. Your isolation of a sample should incorporate a purification kit that will produce a relatively pure sample.

• 7

  Review literature or the written protocol to determine if the proper PAGE was used to conduct the experiment. The percentage of acrylamide in PAGE is specific to the size of the sample to be tested. The literature could also provide specific information with respect to set up of the power supply and rate of sample migration.