LDH Purification lab Report
PURIFICATION OF LACTATE DEHYDROGENASE FROM CHICKEN MUSCLE TISSUE
Abstract
The enzyme lactate dehydrogenase (LDH) catalyzes the last step of anaerobic glycolysis that is important for the normal function of the body. Purification of LDH is essential to understand its structure and function. The purpose of this experiment was to extract and purify LDH enzyme from chicken muscle tissue using a variety of various. Analytical methods such as activity and protein assay were employed to determine the presence and purity of LDH. The cells were initially disrupted and proteins were solubilized. LDH was purified from the ammonium sulfate precipitated protein mixture by affinity chromatography and its activity was studied by spectrophotometric determination of NADH at 340 nm. From Pierce BCA assay of crude homogenate, initial protein concentration was shown to be 100 mg/ml. The final protein concentration of the pooled affinity sample was shown to be 0.2 mg/ml. It was found that the total specific activity of LDH was 58.5 µmol/min/mg, and yield of 0.6%. Even though we were successful in purifying LDH enzyme, further steps can be taken to increase the yield.
Materials and Methods Cell Lysis and Extraction of LDH: Approximately 40 g of minced chicken breast meat (40.327 g) is blended with 75ml cold extraction buffer in four 30-seconds bursts for homogenation of the muscle tissue. The extraction buffer contained 10mM Tris-HCl (pH-7.4), 1mM 2-Mercaptoethanol, 1mM Phenylmethylsulfonylflouride (PMSF), 1mM Ethylene diamine tetraacetic acid (EDTA). The homogenization procedure was carried out in the cold room to prevent the denaturation of proteins. The homogenate was centrifuged at 15,000 rpm for 20 minutes at 40 C. The supernatant was filtered through two layers of cheesecloth to remove lipids from the supernatant. The total volume was noted and three 0.5 ml aliquots (crude extract) were stored at -200 C. Ammonium sulfate precipitation: 60% ammonium sulfate
Abstract
The enzyme lactate dehydrogenase (LDH) catalyzes the last step of anaerobic glycolysis that is important for the normal function of the body. Purification of LDH is essential to understand its structure and function. The purpose of this experiment was to extract and purify LDH enzyme from chicken muscle tissue using a variety of various. Analytical methods such as activity and protein assay were employed to determine the presence and purity of LDH. The cells were initially disrupted and proteins were solubilized. LDH was purified from the ammonium sulfate precipitated protein mixture by affinity chromatography and its activity was studied by spectrophotometric determination of NADH at 340 nm. From Pierce BCA assay of crude homogenate, initial protein concentration was shown to be 100 mg/ml. The final protein concentration of the pooled affinity sample was shown to be 0.2 mg/ml. It was found that the total specific activity of LDH was 58.5 µmol/min/mg, and yield of 0.6%. Even though we were successful in purifying LDH enzyme, further steps can be taken to increase the yield.
Materials and Methods Cell Lysis and Extraction of LDH: Approximately 40 g of minced chicken breast meat (40.327 g) is blended with 75ml cold extraction buffer in four 30-seconds bursts for homogenation of the muscle tissue. The extraction buffer contained 10mM Tris-HCl (pH-7.4), 1mM 2-Mercaptoethanol, 1mM Phenylmethylsulfonylflouride (PMSF), 1mM Ethylene diamine tetraacetic acid (EDTA). The homogenization procedure was carried out in the cold room to prevent the denaturation of proteins. The homogenate was centrifuged at 15,000 rpm for 20 minutes at 40 C. The supernatant was filtered through two layers of cheesecloth to remove lipids from the supernatant. The total volume was noted and three 0.5 ml aliquots (crude extract) were stored at -200 C. Ammonium sulfate precipitation: 60% ammonium sulfate