CSUMB Bio 310L - Crude Mitochondrial Preparation from Mussel Gill

Lab #4 The objective of the first week of this lab is to: Prepare a crude mitochondrial membrane fraction from Mytilus spp. gill tissue.       During the first period of this lab students remove gill tissue from Mytilus spp. (an intertidal mussel) and prepare a homogenate for centrifugation differential centrifugation. Students will collect fractions at each step of the preparation. After a low speed and a high speed spin, they freeze the pellet. This is their crude mitochondrial membrane fraction. During the second lab period students assay the ATP hydrolysis activity of the fractions they collected. During the third lab period students assay the protein concentrations of the fractions they collected. This permits them to calculate the specific activity of each of their fractions. During the fourth lab period students conduct SDS-PAGE of their fractions. They also use software to analyze the results of their assays. During the final lab students analyze their gels and compare the results to known proteins in the mitochondrial membrane. Preparation of crude mitochondrial preparation from mussel gill tissue (Bio 310) Collect Bay Mussels (Mytilus spp.) from under floating docks or California Mussels (Mytilus californianus) from the rocky intertidal (fishing licence or collecting permit required) and store in 15 º (±3º) C seawater. Identify and know how to use the equipment at your station. Prepare a flow chart of the steps in the procedure using the instructions below. Label the tubes you will need ahead of time and write the description of the labels in your labbook. Use a flat, blunt blade or butter knife to pry open the shell enough so that you can see the posterior adductor muscle (see attached diagram). Sever this muscle with a scalpel and then the other smaller muscles. Re-immerse the two mussel halves in seawater (small finger bowl). Using a forceps and scissors, retract and clip out the ctenidia (gills) from 1 bay mussel. Rinse well in the seawater. Then rinse (dip and wash) the tissue in Homogenization Buffer (HB, see below). Using a scalpel or a single edge razor blade, mince about 0.1 grams of gill tissue very finely (at least down to 1 mm cubes) on a glass microscope slide. About 10 cm2 will usually be plenty and fits on one microscope slide. Wash the 0.1 grams of minced tissue into the class beaker using 2 mL ice cold HB.  The instructor will homogenize the pooled minced tissue in 10 - 15 mL HB with at least 10 strokes loose and 10 strokes tight in the Dounce Homogenizer and accumulate the homogenate in a gently stirring beaker on ice. Fill two 1.6 mL flip-top eppendorf tubes with 1.0 mL of the gently stirring homogenate. After vortexing the two tubes remove a 50 uL sample from each and freeze in a labeled tube. Bring the 10 tubes (2 from each group) up to 3,000 x g for 30 seconds and allow to spin down without braking. Remove a 100 uL sample of the supernatant and freeze. Carefully remove the rest of the supernatant to a fresh tube and spin at 18,000 x g for 10 minutes. While those tubes are spinning, resuspend one pellet in 1.0 mL ice cold HB, label, and freeze. Resuspend the other in 1.0 mL ice cold Resuspension Buffer (RB, see below), label, and freeze. When the tubes are finished with the 18,000 x g spin, carefully remove the supernatant to a tube, label, and freeze.  The final pellet should be resuspended in 1 mL RB and frozen.       Homogenization Buffer (HB) 0.5 M sucrose 0.15 M KCl 70 mM Tris 0.1 % BSA (low fat) (1g/1L) adjust to pH 7.5 with HCl. 10 mM Tris-EGTA 10 mM Tris-EDTA adjust again to pH 7.5 with HCl. Add these two just prior to use: 1 mM Dithiothreitol 0.1 mM PMSF       Resuspension Buffer (RB) 0.5 M sucrose 0.15 M KCl 50 mM Tris-HCl 1 mM Tris-EDTA pH 8.0 adjust to pH 7.5 with HCl. Add the DTT just prior to use: 1 mM Dithiothreitol