Chapter5:
Procedure 5.2

Polyacrylamide Gel Electrophoresis

Background

Running polyacrylamide gels is part of working in any modern laboratory where protein or nucleic acid samples are analyzed. Typically, when purity needs to be monitored closely, for example, when proteins are being purified for the first time, PAGE is used before and after each purification step to evaluate the success of that step. We will use it to evaluate the success of our purification by running samples on the gel from each stage of our purification. PAGE is an indispensable analytical tool for protein chemists and there is no other technique with the resolving power of PAGE that is as inexpensive and widely available.

SDS Polyacrylamide gels (SDS-PAGE) that we will be using in this procedure, are called “denaturing gels” because they contain sodium dodecyl sulfate (SDS), an ionic detergent that binds to the amino acid residues in the proteins. Due to its ionic properties, SDS confers a net negative charge on all the proteins, overcoming any intrinsic charge; in this way the proteins uniformly migrate toward the positive electrode. SDS also disrupts the secondary and tertiary structure of the proteins, essentially destroying their globular configuration and making them into linear molecules that then migrate in the electric field on the basis of their size. PAGE is a very powerful technique because even small differences in molecular weights produce distinguishable bands on a gel.

REMEMBER THAT ACRYLAMIDE IS A POTENT NEUROTOXIN. ALWAYS WEAR GLOVES WHEN WORKING WITH IT.

There are many different types and brands of electrophoresis equipment. There are also many different methods for casting gels, including commercially prepared gels that are purchased “ready to use”. As you encounter these in the workplace, remember that none of these methods is necessarily better or worse although some methods are easier than others. They all accomplish the same goals.

Before you begin, make sure you understand the basic principles of SDS-PAGE as discussed in lecture. In addition, read all the instructions and watch the instructor’s demonstration before attempting this procedure. Bear in mind that there is no substitute for repetition. The more gels you run, the better your gels will turn out.

You will prepare and run at least three gels. The first gel will be a practice gel. For the practice gel choose samples that are not scarce or valuable such as the commercial ß-galactosidase and the molecular weight markers. Do not use samples from your purification until the second gel. Hopefully, you will make all the mistakes you are going to make on the first gel and won’t have to waste your valuable samples.

For the second gel, you will need the following samples:

• pure ß-galactosidase (commercial preparation)
• crude extract
• dialysate or column start (sample you loaded on your column)
• pooled samples from your column
• any other samples you wish to run

For the first and second gels, protein bands will be detected using Coomassie Blue staining. The third gel will not be stained and be used for the immunoblotting procedure, which is discussed later. Additional gels can be run for silver staining or other staining methods if time permits.

Preparing your samples:

Note that the sample buffer has ß-mercaptoethanol in it. Handle samples in the fume hood if possible.

1. Prepare your samples. Place 18 µl of each sample in a microfuge tube and add 6 µl of sample buffer (4x) to each sample. If you have less than 24 µl of a sample, make up the difference with water. These instructions are based on a 10 well comb and a 1.5 mm thick gel. Note that the well volumes formed by different combs and different gel thickness accommodate different sample volumes. Refer to Table 5.1 for details.
2. Using a pushpin, poke a hole in the top of the sealed microfuge tubes containing your samples. Place the tube in a boiling water bath for 2 minutes. After removing the tubes from the boiling water, spin down any condensed water or disturbed sample in a microfuge for a few seconds. Boiling your samples in sample buffer changes your proteins into long strands, disrupting their three-dimensional structure and making all the amino acids available to bind the negatively charged detergent, SDS. Note that the sample buffer contains ß-mercaptoethanol (ß-ME).
3. Check to see that each well has roughly the same volume; in this case, approximately 25 µl. Use a gel loader tip to carefully layer each sample in its well. The sample will sink to the bottom of the well, displacing the buffer. It is usually a good idea not to use the first and last wells.

• Place the safety lid on the unit and attach the leads to the power supply.

Loading the gel:

1. Check for availability of all needed reagents and equipment. See Appendix A for recipes.
   
   • Sample buffer
   • Running buffer (Laemli buffer)
   • Staining solution or fixative (depending on visualization method)
   • Apparatus
   • Precast gels (make sure they fit with your apparatus and do not leak)
   • Power supply
2. Remove pre-cast gels from packaging and assemble in apparatus according to manufacturer directions (instructor hand-out optional here).
   Gently remove the comb. Rinse the sample wells with running buffer and drain by inverting and shaking the unit over the sink.
3. To facilitate sample loading, a ‘well location decal’ can be used. The decal is a clear plastic square that adheres to glass when wet. The outline of three different combs, corresponding to 5, 10 and 15 wells respectively, is marked along three separate edges of the decal. Simply wet the decal and place it against the front of the sandwich with the appropriate edge outlining the sample wells. Alternatively, you can take a marker and put a dot under each of the wells.
4. Fill the sample wells and the upper and lower buffer chambers with electrophoresis buffer (Laemli buffer).
5. Using a micropipettor (extra long tips can be used), carefully load each well with the appropriate sample. DO NOT forget to write down the contents of each well.
   

SAFETY NOTE: The electrophoresis unit is designed to prevent you from contacting the gel or buffer while voltage is applied. Never by-pass this safety feature. Also, never touch a puddle around a gel box while voltage is applied to the apparatus.

Running the Gel

Gels may be run at either constant current or constant voltage. We are using what is known as the discontinuous (Laemmli) system because the upper and lower gel buffers are different. Constant current is usually used with a discontinuous buffer system so that the rate of electrophoretic migration will remain constant throughout the run. We have obtained good results at a constant current of running 15-20 mA. per gel. If you have two gels, you will need to double the mA. However, we have also found it convenient to run Laemmli gels at constant voltage of 90 V –125 volts using a small power supply such as the Hoeffer PS 150. (Although you do double the current if running two gels, do NOT double the volts for 2 gels. Why?) Refer to the product literature for the power supply for help. (What might happen if you run your gel at too low a voltage or amperage? What might happen if you turn it up too high?)

1. Place the safety lid on the gel box unit and attach the electrode leads to the power supply. Turn on the power to the power supply and set it to the appropriate voltage or current level. Check to make sure that bubbles are forming around the electrode wires.
2. Periodically check your gel to make sure the running buffer has not leaked out of the upper buffer chamber. This is especially important when using precast gels from some manufacturers. (What will happen if the upper chamber is empty?)
3. When the tracking dye reaches the bottom of the gel, i.e., below the surface of the lower buffer, turn off the power supply, disconnect the leads and remove the lid of the unit. Do not run the dye front off the gel.
4. Pour out the buffer by inverting the entire unit over a sink.
5. Pry open the gel sandwich. Remove the spacers and peel the gel off the plate into a tray of stain. Wetting the gel helps to loosen it from the plastic plate.
6. Rinse the lower buffer chamber and upper buffer chamber pods with distilled water after each use. Be careful not to damage the platinum wires in your electrophoresis apparatus. Be sure to clean everything carefully and put all the parts away properly.
   

Contact Us:

Lisa Seidman
lseidman@matcmadison.edu
(608) 246-6204

Jeanette Mowery
jmowery@matcmadison.edu
(608) 243-4307