Pig heart instructions and Preparation of Crude Extract and 20,000 X G 2014(1)

Pig heart instructions and Preparation of Crude Extract and 20,000 X G 2014(1)

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Bovine LDH Purification Experimental Procedures Materials: 100 g Beef Heart (provided by instructor) LDH Kinetic Assay buffer, 0.15 M CAPS, pH 10.5 Lactate, 150 mM 45g Ammonium sulfate (pulverized with a sterile mortar and pestle) Homogenization buffer (0.05 M sodium phosphate, pH 7.0) Q-Sepharose® buffer, 0.03 M bicine, pH 8.5 (for dialysis following salt fractionation and IEX chrom.) 2 boxes of sterile 200 µl pipette tips 2 boxes of sterile 1 ml pipette tips Sterile cheesecloth Sterile, large, glass funnel Sterile, 30 ml Corex tubes Sterile, 15 ml Corex tubes Methods All of the following procedures should be done with care to make sure that the LDH fractions are not contaminated. 1. Use of sterile glassware and the wearing of gloves will help. 2. Doing the procedures in a cold room or on ice is required for a successful purification. 3. The class will begin by homogenizing 100 g of bovine cardiac muscle into 400 ml of cold homogenization buffer. 4. Each team will begin the purification scheme with 100 mL of crude homogenate (crude extract). We have fourteen 30 ml Corex centrifuge tubes so the centrifugation steps will be done as a group to account for the number of tubes and rotor spaces. 5. At each step of purification, it is important to record the volume of the sample you are working on. 6. Save 1.5 to 2.0 mL in 500 μl aliquots at -20 C 0 C for kinetic assays, BCA protein assays, and SDS-PAGE. Preparation of LDH Crude Extract (initial fraction) from Bovine Heart Tissue 1. Remove the obvious fatty deposits and connective tissue with a scalpel. 2. Cut up the bovine (beef) heart into ½ inch pieces. 3. Combine 100g of bovine heart with 300 mL of cold 0.05 M potassium phosphate, pH 7.0 4. Add 1 ml of Sigma Protease inhibitor cocktail for mammalian tissues per 20g of heart tissue. 5. Homogenize at high speed in the blender for 2-3 min at 4°C. 6. Filter the crude homogenate through sterile cheese cloth into a sterile beaker or bottle to remove larger debris.
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