MEDBEd: Teachers Prep for Proteomics 12-3-10

_____ 1. MEDBEd would like to help you at your school. If you would like Donna and Donita to travel to your school to help prepare for the lab or to help teach during classtime, please email MEDBEd at or call the MEDBEd office at 918-595-4611.

_____ 2. Excluding any prelab/postlab activities, gel drying, and discussion, the wet lab part of the activity is normally a 2 period or 2 day lab activity. Please plan accordingly.

_____ 3. Gather together the following documents and make copies as needed:

A.Pre-tests and Post-tests (MEDBEd provided 60 of each of these with large clasp envelopes addressed to TCC. Please print more as needed. The students will take the pre-test BEFORE they see the manuals and BEFORE any pre-lab instructions. Remind the students that they are not expected to do well on the pre-tests and that the same questions will reappear later as a post-test after the lab is completed.There are no student pre and post tests for the teachers who attended the SEEDBEd GAP workshop. Until June of 2011, MEDBEd teachers should use the MEDBEd Pre and Post tests and return them to TCC.)

B.Instructor’s manual for the Bio-Rad “Biotechnology Explorer, Comparative Proteomics Kit 1: Protein Profiler Module. (This is the manual that came with your Proteomics kits at the MEDBEd Workshop #3. In addition to basic lab prep instructions, the manual includes good background information.)

C.List of “Proteomics Equipment to be Given to High Schools.” (This is the printed list of free items which were given to you at the workshop. The second page of that document also lists some items that need to be provided by your school. On pages 28 – 32, the Bio-Rad instructor’s manual provides similar equipment lists.)

D.Student instruction manuals(The Bio-Rad Student Manual begins on page 47 of the Bio-Rad Instructor’s Manual. In addition to lab instructions, the student manual also includes some good background information. Choose the pages you wishto duplicate for your students.)

E.TCC Model Release (Make copies of this document if you wish to share photos of your students.)

_____ 4. Go to Bio-Rad.com tosee the power point teaching resources for Proteomics. At the far right, choose the “Life Science Education” option. Then select “About the Program” and “Teaching Resources.” Scroll down until you see “PowerPoint Presentations for Workshops and for Teaching.” Select “Comparative Proteomics Kit I: Protein Profiler Module.”You may wish to use some of the power point slides with your students. (Please note that Bio-Rad also has PowerPoint Presentations for ELISA HIV, PV92 PCR, and Chromatography. Bio-Rad does not list a PowerPoint Presentation for Precut Lambda, but some parts of the PowerPoint forthe “Forensic DNA Fingerprinting Kit” may be helpful.)

_____ 5. Gather together the necessary equipment listed on the document, “Proteomics Equipment to be Given to High Schools.” (See item #3C above.) We removed the Bio-Rad proteomics kits from their green cardboard boxes and placed all of the proteomics supplies together into one of the large plastic tubs.

_____6. Prepare the TGS buffer. Label an empty gallon size distilled water jug, “1x TGS” with date. To make 3700 mLof 1x TGS buffer, mix 370 mL of 10x TGS with 3.33 L (3,330 mL) of DI water or distilled water. Each vertical electrophoresis chamber requires about 900 mL of buffer. Store the TGS buffer at room temperature. Any TGS buffer remaining in the chamber after electrophoresis, should be rinsed down the drain with lots of water.

_____ 7. Buy small samples of 5 to 8 different kinds of raw fish from the grocery store. At TCC, we use catfish, salmon, pollock, snapper, tuna, halibut, scallop, and shrimp. Each fish sample should be a cubic inch or smaller. Ask the butcher to write the variety of fish on the butcher paper as he/she wraps each sample. Store the samples in the freezer. With a sharp scalpel, it is easy to cut off the small pieces of frozen fish as needed for Lesson #1. Refreeze the remaining fish for future use.

_____ 8. Prepare 71% alcohol to be used for cleaning work areas. Mix 390 mL of 91% isopropol alcohol with 110 mL of DI water or distilled water. (If you prefer to purchase premixed alcohol, both 70% and 91% concentrations are readily available at your local pharmacy.)

_____ 9. Check the supply of micropipette tips. The instructor will need the yellow BR-35 tips. Students will use only the long thin protein electrophoresis tips to load the vertical gels.

_____ 10. Final set up for Day #1/Lesson #1 Lab:

  1. See the “Instructor’s Advance Preparation” directions in your instructor’s manual, page 28. You will not use DTT. MEDBEd removed the DTT from your proteomics kits.
  2. You will not be using the vertical electrophoresis chambers in lesson #1. However you may wish to consider the following information to help you decide how many teams you will want per class for lessons #1 and #2: Because MEDBEd provided only 3 vertical electrophoresis chambers, you may wish to divide each class into 3 to 6 teams. With six teams, each chamber would be shared between two teams. However each team would have their own gel because each vertical chamber can accommodate 2 gel cassettes. Simply remove and replace the dam with the second gel cassette.
  3. Bring your water bath temperature to 95°C. It may take several hours to reach the correct temperature, so allow plenty time. Depending on the number of teams using the water bath at one time, you may need to create a second water bath by using your hot plate, thermometer, and a beaker/pan. If you are using up to 6 fish samples per team, you will need enough floating space in your water bath for 1 foam test tube holder per team. If you are using 7 or 8 fish samples per team, you need enough floating space in the bath for 2 foam floaties per team.
  4. Set up student workstations as listed on page 29.
  5. Refer to the Bio-Rad Student Manual, pages 64-66,for the Lesson #1 lab procedures.
  6. When Lesson #1 is completed, store the fish protein samples in the refrigerator if they will be used within a week. Store them in the freezer for extended periods of time.

_____ 11. Final set up for Day #2/Lesson #2 Lab:

  1. See the “Instructor’s Advance Preparation” directions on page 28 in your instructor’s manual.
  2. Refer to the Bio-Rad Student Manual pages 67 to 70 for the Lesson #2 lab procedures.
  3. For the teacher’s prep of the Actin & Myosin standard, you will need a water bath at 95°C. See notes in #9c above.
  4. Set up 3 to6 student workstations with materials listed on page31 in the Instructor’s Manual.
  5. The PAGE gels provided for you by MEDBEd are gels with a one year shelf life. Please check the expiration date prior to your use. Bio-Rad also sells PAGE gels with a 3 month shelf life that are a little less expensive. Both will work well with this lab. The gels need to be refrigerated until ready to use.
  6. Some of the Proteomics Instructor’s Manuals still show diagrams of an older version ofvertical electrophoresis chambers which are no longer available. The vertical chambers that MEDBEd provided to you are a newer version and a little easier to use. After removing the plastic cover surrounding the gel cassette, pour off the excess liquid, remove the green tape along the bottom of the cassette, and remove the green comb from the top of the gel cassette. Remember to place the gel cassettes into the chamber with the shorter side of each cassette nearest the center of the inner chamber/module. You will need to completely fill the inner chamber/module with the 1X TGS buffer and make sure to cover the short plate. Also fill the outer tank chambers with buffer. Refer to the instruction manual that came with your electrophoresis chamber or you can find that same document on the Bio-Rad website at:
  1. Since the students would benefit from a teacher demonstration on how to load the vertical gels during lesson #2, Dr. Spencer recommends that teachers plan to load the “Precision Plus Kaleidoscope prestained protein standard” into the gel for each team. Each kit comes with 50 µL of the Kaleidoscope standard. Divide that amount equally among your teams. Be sure to use only the long skinny protein tips. Demonstrate loading the micropipette very slowly for accuracy in measurement and to avoid bubbles. Then place the protein tip inside the inner chamber/module and against the wall of the cassette nearest you. Pull the tip against the cassette wall and slowlyupward. You will feel and/or hear the tip as it reaches the loading area lip. Demonstrate loading the gels very slowly. Fast loading and/or air bubbles in the well will push the protein upward and out of the well.
  2. When the students are finished loading, you are ready to run the gels. Please use the following procedure sequence:

_____ Please do not allow the students to touch your power supply! Your power supply came with an instruction manual. Please refer to that manual or you can find the same document on the Bio-Rad website at:

_____ Pusheachchamber lid downward until it fits snuggly in place.

_____ With the power supply unplugged, the instructor will insert the electrical leads of each

chamber into the power supply outlets, matching red to red and black to black. All three chamberscan be connected to the power supply at one time.

_____ Plug the power supply into a properly grounded electrical outlet.

_____ Turn on the power switch located on the right side of the unit near the back.

_____ Select the “constant” key at the lower right front.

_____ Push “V” to select constant voltage until the LED light above the “V” lights up.

_____ Push the “+/-“ scroll key until the display shows “200” for 200 volts.

_____ To start the run, push the run/pause key until the LED lights up above the running symbol.

_____ After 35 minutes, stop the run by pushing the stop key.

_____ Turn off the power switch found on the right side of the machine near the back.

_____ Unplug the power supply from the electrical outlet.

_____ Unplug the electrophoresis chamber power leads from the power supply unit.

12. After running the gels, you are ready to stain and destain the gels.

_____ Turn off the power supply as instructed above.

_____ Pour off and discard the running buffer. Rinse the buffer down the drain with lots of water.

_____ Remove the gel from the chamber. DO NOT THROW AWAY THE DAMS!

_____ Rinse the chamber tank, module, and dams with distilled water.

_____ Rinse the gels several times with tap water in the staining tray for up to 5 minutes.

_____ Pour off water from staining trays. Replace with 25 mL Bio-Safe Coomassie stain per gel.

_____ Allow the gels to stain for 1 hour. Occasionally shake gently.

_____ After 1 hour pour off the stain. Rinse it down the drain with lots of water.

_____ While still in the staining tray, cover the gel with a large amount of water and allow it to

destain overnight. Occasionally shake gently if possible.

_____ 13. Final set upfor Day #3/Lesson #3 Lab:

MEDBEd does not supply the “GelAir cellophane support sheets,” the “GelAir assembly table,” or the

“GelAir drying frame” mentioned in your BioRad instructor’s manual. Alternate instructions for drying

gels, will be given during the MEDBEd workshop.