Teacher Guide: Muscle Protein Electrophoresis Experiment

The BIOTECH Project will provide all of the materials and supplies you will need to conduct this experiment, unless otherwise noted. The materials and procedure necessary for conducting the experiment are outlined below. Trouble-shooting and practical advice is included; hopefully it will be helpful. Please feel free to contact the BIOTECH Project with any questions or suggestions.

This guide is provided for your reference, and the BIOTECH Project does not provide any additional handouts, worksheets, or assessment tools for the students. Feel free to cut and paste from this experiment description and/or develop your own handouts or assessments. If you're willing to share the materials you develop, they will be used to improve the experiment, share with other teachers, and document your efforts for BIOTECH Project fund-raising.

BIG IDEA: Although all muscle types (skeletal, smooth, and cardiac) have both actin and myosin that help them contract, the mechanism of contraction is different: cardiac and skeletal muscle use tropomyosin and smooth muscle doesn't. Since smooth muscle doesn't need tropomyosin to be able to contract, it doesn't make the tropomyosin protein. During this experiment, we will separate out the muscle proteins from cardiac, skeletal, and smooth muscle using protein gel electrophoresis. We will compare the muscle proteins to actin, myosin, and tropomyosin protein controls and determine which proteins each muscle type makes.

Materials and Procedure for Muscle Protein Electrophoresis

Materials

• 1.2% agarose in Tris-Glycine buffer (30 mls per group)
• Tris-Glycine-SDS buffer (250 mls per group, can be reused 5 times)
• Coomassie Blue stain (Coomassie Brilliant Blue, acetic acid, methanol, water; 20 mls per group, can be reused 5 times)
• Destain (acetic acid, methanol, water; at least 50 mls per group, cannot be reused)
• Muscle samples (smooth, cardiac, skeletal)
• Sample buffer for each muscle sample (bromophenol blue, SDS, Tris, glycerol in microfuge tubes; 3 tubes per group)
• Actin, myosin, tropomyosin samples (protein controls; 1 of each per group)
• Dithiothreitol (DTT) - This is a poisonous chemical, do not get it on your skin.
• Gel electrophoresis trays
• Power supplies and wires
• Pipettes and tips
• Gel holders for staining
• Saran wrap
• Sharpie pens
• Light boxes and power cords
• Water bath (set to 55ˇC)
• Spatula
• Heat block (set to 95ˇC)
• Microfuge tubes and rack
• Pair of gloves (use when working with DTT)

Materials not provided

Microwave

Paper towels, kleenex, or kimwipes

Procedure for running protein gels

1. Get your electrophoresis apparatus. Make sure one comb is in place at the negative electrode (black end of the gel). If you'd like students to practice pipetting at the other end of the gel (red end, positive electrode), have them put both combs in place at either end of the gel. Make sure that there are stoppers at both ends of the gel space.

   Eventually, the students will load their protein samples in the wells at the negative end of the gel. The proteins will then migrate toward the positive end because they are negatively charged. If students accidentally plug the electrodes in backward or load into the red/positive electrode wells, their samples will migrate off the gel into the buffer instead of into the gel.

2. Pour hot agarose into the gel space until it reaches the top of the gel box. Let the agarose harden, which should take about 10 minutes. DonŐt touch/move your gel until itŐs hard.

   While the gels are hardening, the protein samples can be prepared, if they have not been prepared already.

3. When the agarose gel is hard, take out the stoppers and pour Tris-Glycine-SDS buffer over your gel so that is it completely covered plus a little more.

   Tris is a buffer that controls the pH during electrophoresis. The process of electrophoresis involves an oxidation/reduction reaction (formation of H2 gas at the negative electrode and O2 gas at the positive electrode) that alters the pH at each electrode. The Tris buffer helps minimize that. The SDS is a detergent that denatures and sticks to proteins, keeping them unfolded and negatively charged during electrophoresis.

4. Draw a diagram of the gel including the wells. Label which protein samples you are putting into which wells. Once the protein samples are loaded, you will not be able to determine which is which.

5. Run that gel!! Plug the electrodes into your gel box (red to red, black to black), being careful not to bump your gel too much. Plug the power source into an outlet and set the voltage to 100 V.

   You can tell the gel is plugged in and running when you see the bubbles of H2 and O2 gas forming at the electrodes. Eventually, you will see the dye in the protein samples moving toward the red/positive electrode because it too is negatively charged.

6. Let the gel run until the dye migrates 2/3 of the distance of the gel (about 30 minutes). Now your gel is ready to be stained. You can stain it directly or store it overnight in the refrigerator wrapped in saran wrap (gels will keep even over a weekend).

7. When you're ready to stain, put gel in gel holder for staining. Pour just enough stain over the gel to cover it. Cover the holder with saran wrap and allow to stain for at least 30 minutes, periodically rocking gently. [The gels can stain for longer, but the longer they stain, the longer they will take to destain.]

8. Pour stain back into stain bottle (stain can be reused for all your classes during the day). Pour enough destain on the gel to cover it. Tuck a paper towel or some kleenex in the holder with the gel (it will soak up the stain). Cover with saran wrap and allow to destain for at least 2 hours and up to several days. Usually overnight is long enough to readily view the protein bands.

9. Put the gel holder with the gel in it on the light box and view your gel.

Procedure for preparing muscle samples

1. Each group needs to assemble their muscle samples and the protein controls (actin, myosin, tropomyosin). Each group should collect 3 sample buffer tubes and label one tube 'skeletal', one tube 'cardiac', and one tube 'smooth'. Cut a small bit of muscle and put it in each tube.

   The sample buffer includes bromophenol blue (allows you to see the sample while you're loading it), glycerol (which is dense and helps the samples sink to the bottom of the wells), SDS (a detergent than denatures and negatively charges proteins), and Tris (a pH buffer).

2. Add tiny pinch of DTT into the 3 muscle sample tubes. Gently shake tubes and let samples sit for 5 minutes. In the meantime, get 3 new microfuge tubes and label them 'skeletal', 'smooth', and 'cardiac'.

   DTT denatures proteins by breaking disulfide bonds, helping unfold the proteins so they can move freely through the agarose during electrophoresis. Thus, the proteins will separate by molecular weight and charge, and not by 3-dimensional structure.

3. Pipette a quarter of the liquid (not the muscle) from your sample tubes to the new tubes. Incubate all 6 tubes in the heat block at 95ˇC for 5 minutes (cardiac, skeletal, smooth, actin, myosin, tropomyosin).

   Heat also helps denature the proteins.

4. Samples are ready to load into the gels. Be sure to keep track of which samples are loaded into which wells.

   
   
   

   BIOTECH Home | Laboratory Activities | Biotechnology Resources |
   On Tour with BIOTECH | BIOTECH Bulletin Board |

   

   BIOTECH Project Department of Molecular and Cellular Biology The University of Arizona September 14, 2000 Nadja Wehmeyer, Ph.D. nadja@email.arizona.edu http://biotech.biology.arizona.edu