Preparation of Your Own Samples |
Teacher's Note:
Other fish samples can be obtained from your local fish store, pond, or lake to make gel comparisons and to determine evolutionary relationships among various fish samples. Students also may investigate commercial or popular fish foods. For example, shark plugs (fin meat), treated with squalene oils and polysaccharides often are substituted for large bay scallops. Artificially flavored crab meat is pollack that has been chemically and mechanically treated to resemble crab legs. Electrophoresis can be used to distinguish the real sample from its artificial substitute. Use the following protocol for sample preparation.
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Tris-HCl-SDS Solubilization Buffer
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25 mL for SDS gels (Tris-SDS) 36W5292
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Buffer
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Composition (g/L)
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Molarity or
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%
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Tris
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15.16 g /L
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0.125
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M
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SDS
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20
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2.0
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%
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Sucrose
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100
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10.0
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%
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Bromophenol Blue
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0.1
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0.01
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%
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Treated to pH 8.0 with concentrated HCl
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Preparation of Animal Muscle Sample:
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(a)
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Use 1 g of animal muscle tissue (enough for 1-2 gels). Be sure to remove any skin or bone if you are using fish.
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(b)
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Dice the tissue to very fine pieces with a razor blade or blend it. The sample also can be pulverized with a mortar and pestle.
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(c)
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Place the blended sample in a small beaker and add 2 mL of distilled or deionized water.
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(d)
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Add 3 mL of the solubilization buffer and stir vigorously.
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(e)
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Pour the sample into a small test tube and let the undissolved solid muscle matter settle at the bottom. This will take approximately 30 minutes. To speed up the process you may wish to centrifuge the sample for 10 - 15 minutes.
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(f)
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After settling or centrifuging, save the dark blue liquid extract (top layer) in separate vials. Discard the solid matter.
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(g)
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Incubate the sample for 2 minutes at about 9
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(h)
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After incubation, filter the sample by pouring it into a filter-lined funnel. Filter until the mixture has gone completely through the filter. This may take 10 to 15 minutes. Your sample is ready to be electrophoresed.
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(3)
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In this experiment 3 fish muscle samples will be electrophoresed along with a molecular weight protein marker. It is suggested that each student team receive its own set of samples prepared in advance by the instructor.
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Label student microcentrifuge "reaction" tubes (set of 4 per student group) as follows:
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Tube # 1 - Yellow Perch
Tube # 2 - Walleye
Tube # 3 - Chinook salmon
Tube # 4 - High molecular weight protein marker
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(4)
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Using a 20 uL micropipette (see "Micromeasurement Section" for micropipetting tips), transfer 20 uL of each of the above sample types into corresponding student reaction tubes. (Use the microcentrifuge tubes provided.) Cap the tubes. Note: Any unused samples should be stored in a refrigerator.
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(5)
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Use the enclosed Ward's copymasters ("Student Study" and "Student Analysis") to make worksheet copies. Permission is granted for unlimited copies to be made for use within a single school building.
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Additional Preparation Notes:
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You may wish to designate one area of the laboratory as an "electrophoresis area" where cells and power supplies are connected. Assure that any wall outlet is properly wired, i.e., that correct polarity exists (use a circuit tester). Assure that electrical power cords and patch cords are clear from moisture or wetness.
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Assure that all students are thoroughly drilled in the correct procedure for making electrical connections and that they are directly supervised by you.
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Assure that all students wear the following personal protective equipment: Safety goggles, smock or apron (while loading gels and during electrophoresis) and protective gloves (during staining).
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AE Partners Collection Index
