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BCH102 Lab Manual

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Lab Manual for BCH102 at UCR
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  BCH 102 Laboratory Manual M-1 Introductory Biochemistry Laboratory Manual   BCH 102  BCH 102 Laboratory Manual M-2 Table of contents Experiment 1 Purification of native LDH from chicken ........................................................... M-3 Experiment 1A:  Buffer and Column preparation ..................................................................... M-5 Experiment 1B:  LDH extraction and precipitation ................................................................... M-7 Experiment 1C:  Pseudo-affinity chromatography .................................................................. M-11 Experiment 1D:  Determining Protein Concentration .............................................................. M-15 Experiment 2 Purification of recombinant lactate-dehydrogenase from E. Coli by metalion affinity-chromatography .......................................... M-18 (Lab2A-D:  Important safety instructions .......................................................................... M-24) Experiment 2A:  Buffer preparation and bacteria inoculation ................................................. M-25  Experiment 2B:  Protein Expression ........................................................................................ M-28 Experiment   2C:  Purification of (His)LDH by Nickel-bead affinity chromatography ............ M-30 Experiment 2D:  Determining Protein Concentration .............................................................. M-33 Experiment 3: Analysis of LDH Purity: SDS-Polyacrylamide Gel Electrophoresis SDS-PAGE ............................................................................ M-36 Experiment 3B:  SDS-PAGE Day 9. Destain SDS gels continued .......................................... M-42 Experiment 4 Yield and Specific Activity of LDH ................................................................. M-43 Experiment 5 K  M  and k  cat of uninhibited   LDH (Kinetics) ....................................................... M-48 Experiment 6 K  M  and k  cat of inhibited   LDH ............................................................................ M-50 Experiment 1-7  -   Format for Lab Report ................................................................................ M-52 Experiment 8 Molecular Biology - The transformation, purification and restriction enzyme analysis of plasmid DNA ............................................. M-57 Experiment 8a:  Culturing of  E. coli  ........................................................................................ M-61 Experiment 8b:  Culturing of  E. coli  ........................................................................................ M-62   Experiment 8c:  Plasmid Purification ...................................................................................... M-65 Experiment 9 Restriction analysis of DNA ............................................................................. M-68 Appendix  .................................................................................................................................. M-72 Lab   Experiment 1  ........................................................................................................... M-72 Lab   Experiment 2  ........................................................................................................... M-79 Lab   Experiment 5, 6, and 7  ............................................................................................ M-81 References  ...................................................................................................................... M-121 Laboratory Report forms Experiments 1-9  ................................................................ M-122  BCH 102 Laboratory Manual M-3 EXPERIMENT 1-7: Isolation and Characterization of Lactate Dehydrogenase. (Adapted in 1996 for use in BCH 102 from a series of experiments developed by Professor Christopher R. Meyer at CSU, Fullerton) Recommended reading: Lehninger Principles of Biochemistry, D.L. Nelson and M.M. Cox, editors, 5th edition, Section 6.3, pages 194-205. This manual, appendices A-D. INTRODUCTION Lactate dehydrogenase (LDH, B.C. 1.1.1.27) catalyzes the NAD(H)-dependent interconversion of lactate and pyruvate: L-lactate + NAD +   ↔  Pyruvate + NADH + H +  This enzyme is found in almost all animal tissues, microorganisms, and plants, and plays a key role in the equilibrium between carbohydrate catabolism and anabolism. For example, the reduction of pyruvate to lactate (the reverse reaction of LDH), which occurs during anaerobic metabolism, is a key step in regenerating NAD +  to allow glycolysis to continue. One of the interesting characteristics of LDH is that it can exist in different forms because it is a tetrameric enzyme with two types of subunits. Depending on the tissue type (i.e., heart muscle versus skeletal muscle) one type of subunit will be more expressed than the other [H subunit, heart ; M subunit, muscle (skeletal muscle)]. The tissue-specific differential expression of the two types of subunits makes the detection and characterization of LDH important in clinical diagnosis of disease state such as heart disease, where heart muscle damage releases H subunits of LDH into the blood. A good method of detection of LDH is to measure its activity - the rate at which substrate is converted to product under a certain set of conditions. An assay of LDH activity can be  performed by measuring the reduction of NAD +  to NADH or by measuring the oxidation of  NADH to NAD + . These measurements are easily performed because NADH absorbs strongly at 340 nm, while NAD +  does not. Thus, one can monitor the progress of the reaction by measuring spectrophotometrically the change in absorbance at 340 nm as a function of time. GENERAL PLAN OF EXPERIMENT You will purify the LDH from chicken breast muscle tissue by I) homogenizing the tissue to release the soluble enzyme, 2) precipitating the enzyme with ammonium sulfate (this is also a concentration step), 3) absorbing the enzyme to, and subsequently eluting it from, a pseudo affinity column. This column consists of agarose beads to which dye molecules have been attached covalently. These dye molecules structurally resemble purine nucleotides. Therefore, the column absorbs proteins that bind purine nucleotides, allowing them to be separated from other proteins. The bound LDH can be eluted from the column with NAD +  or NADH in moderately high concentrations.  BCH 102 Laboratory Manual M-4 A fairly rapid purification of LDH can be accomplished by this pseudo-affinity chromatography technique. Progress of the purification of LDH will be monitored by measuring  both enzyme activity and protein concentration. You will use your purified LDH in subsequent experiments that involve the kinetic and physical characterization of LDH. However, if your  purification is unsuccessful, then you will be provided with purified LDH for use in the later experiments.
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