Summer Research Program for Science Teachers

 

Brigette Brady

Forest Hills High School, Queens

August 2005

 

 

 

                                                                                            

Are different proteins expressed during different developmental stages of C. elegans?

 

General goals:

 

Learner Outcomes:

            1. Student should be able to prepare a digest of organic material in preparation for protein electrophoresis.

            2. Students should be able to set up a protein electrophoresis unit with a polyacrylimide gel and connect to a power pack.

            3. Students should be able to master use of micropipets, and microcentrifuges.

            4. Students should be aware of safety procedures necessary when doing a gel electrophoresis, ex. wear gloves and goggles at all times.

            5. Students should be able to explain the chemistry behind each of the reactions done, ex. digesting buffers, use of SDS, Coomassie Blue stain.

            6. Students should be able to connect protein structure to what is happening at every step of the procedure.

            7. Students should be able to analyze their gels and make comparisons to the standard run.

 

Introduction:

            Proteins play a vital role in living organisms. They form the structural parts of organisms as well as play a major role in regulating reactions, acting as carriers for specific molecules, acting as defense molecules, and are involved in multitudinous signaling reactions. These are just a few of the functions of proteins. Proteins can have a complex  folded and globular structure, depending on their specific function. We know that proteins are made in ribosomes, however, their origin is in the nuclear DNA. The DNA holds the code for the proteins. Many signals throughout the development of an organism determine which codes will be transcribed and translated into final protein product.

            In this lab we will use a technology called SDS PAGE protein electrophoresis to determine if there are different proteins present at different developmental stages of the organism C. elegans. In electrophoresis the proteins are separated by an electric field based on size (molecular weight).C. elegans has four different larval stages before becoming an adult. Can you make a prediction on whether or not you believe different proteins will be expressed at these different larval stages? Why do you believe this? You will prepare digests of the four larval stages and run them on a gel along with a molecular standard. The standard comes with a chart that indicates the molecular weight of given proteins. You will compare your banding patterns to the standard and to each other to determine if there are different banding patterns.

 

 

Group Instructions:

            Work in teams of three. Read the procedure over with your group. A well organized group will assign work to each member so that the work can be completed in the given amount of time. Example: While one person is preparing the digests, another person can be setting up the electrophoresis unit with the buffer and the gel. Do not handle the polyacrylamide gel or the buffers and stains without gloves and goggles. Remember to label all microtubes, do not rely on your memory! When you complete a step of the procedure, write down what you have done in your notebook.

            This lab can be approached differently, by assigning different groups to one larval stage only. The different groups will then need to share their results with one another for the final analysis.

 

Materials needed by each group:

            For Preparation of lysates

            For SDS Protein Electrophoresis

 

Procedure:

1. One team member should set up the Mini-Protean 3 gel box with the TGS buffer (1x) and pre-cast gel. Make sure that the buffer completely covers the wells on the gel. Another team member should set up a hot water bath; 95C. (These two things can be done while the third team member prepares the worm lysates.)

2. Wash the worm larvae off the culture plates by adding 1 ml (1000microliters) of sterile, distilled, deionized water to the plate. Hold the plate at an angle. As the water flows down the plate suction up the mixture of water and worms back into the pipette. Repeat this action several times to loosen all the worms from the plate.

3. Transfer mix to a 1.5 ml microtube.

4. Centrifuge for 30 -60 sec.

5. Carefully remove microtube from centrifuge without disturbing pellet at bottom.

6. Pipette off the water without disturbing pellet.

7. Add 200 l of Laemmlis buffer to pellet.

8. Flick the microtubes to agitate the tissue in the buffer. Leave at room temperature for 5 minutes with occasional flicking.

9. When the tissue has settled to the bottom, transfer the buffer (without the tissue) into a newly labeled microtube.

10. Repeat steps 2 9 with the other 3 larval stage plates.

11. Place all the labeled microtubes and the Kaleidoscope standard into a holder and place on the 95C hot water bath for 5 minutes.

12. Load your standard and 3 samples into the wells. Record the order of the samples correctly in your notebook. It would be best to load 15 l of the standard followed by 15 l of the L1 sample, then 15 l of the L2 sample, then 15 l of the L3 sample and finally 15 l of the L4 sample.

13. Cover the unit and connect to the power source. Electrophorese for about 30 minutes at 200 volts.

14. When the electrophoresis is complete, switch off the power, remove the lid and place the gel with the plates into distilled water. Remove the remaining plastic around the gel plate and gently remove the gel from the plates. The gel is very thin and can easily break. Wash the gel in another change of distilled water.

15. Transfer the gel into a container of Bio-Safe Coomassie Blue stain. Stain for about 3 hours.

16. After 3 hours, remove the stain wash the gel with 2 changes of distilled water. You may not see bands until the gel has been in the water for an hour or two.

17. You are now ready to analyze your gels.

 

Analysis:

1. Why was it necessary to digest the worms first?

2. What is the function of the SDS in the buffer?

3. What do the banding patterns on the gel tell you?

4. Are there any striking differences in the banding patterns of the different lanes?

5. While it may not be possible to determine the exact identity of a protein, it is possible to estimate the proteins molecular weight. You can generate a standard curve by using semi-log paper, and measuring the distance each of the standard proteins migrated from the well to its final location (on the x axis) against the given molecular weight (in Kilodaltons) on the Y axis. Draw a best-fit line through all the points. It is now possible to determine the molecular weights of all the unknown proteins by measuring the migration distance from the well and drawing a vertical line up to the standard curve. A horizontal line drawn from this point to the Y axis will give an estimate of the molecular weight.

Generate a standard curve and find the molecular weights of some of the dominant protein bands of your sample.

The density of the band is also an indication of the concentration of the protein.

6. Did your results confirm your original prediction about the proteins present at different developmental stages?

7. What questions do you still have about proteins expressed in the different larval stages? Can you think of an experimental design that could be used to answer that question?

 

Going further:

1.You may find that there are several proteins that are seen in each lane. Can you make a prediction about the identity of these proteins? Go to the Wormbase website or the NCBI Website to see if you can make a prediction about some of the actual proteins on your gel based on the molecular weights.

2. Do any of the bands from one lane disappear in another lane? What does that tell you about the genes (DNA) that coded those proteins? What mechanisms exist for turning genes on and off at the level of the chromosomes, at the level of transcription, post-transcriptionally, at the level of translation, and post-translationally?

3. Research some methods; technologies; actually used today for positive identification of specific proteins.

 

Standards addressed:

            1. Teaching Standard A inquiry based instruction.

            2. Content Standard C proteins as components of the cell, the molecular bases of heredity.

            3. Performance Standard 6 Use of Scientific Tools and Technology.