Specific operational procedures for extracting lactate dehydrogenase and its coenzyme using a laboratory centrifuge

Release time:

2014-01-20

Overview:

The materials, reagents, and equipment used for the centrifugal extraction of lactate dehydrogenase and its coenzyme include: (1) animal hind-leg muscle; (2) 9 g/L NaCl solution; (3) activated carbon; (4) 10 g/L lithium lactate solution; (5) 5 g/L KCN solution; (6) mortar and pestle; (7) benchtop centrifuge, etc.

The benchtop centrifuge TD5A-WS can be used for the extraction of lactate dehydrogenase and its coenzyme. Lactate dehydrogenase is an oxygen-independent dehydrogenase that uses coenzyme I (NAD⁺) as a cofactor to catalyze the reversible redox reaction between lactate and pyruvate. Muscles contain abundant lactate dehydrogenase; under conditions of high lactate levels and sufficient oxygen, it catalyzes the conversion of lactate into pyruvate. In in vitro experiments, for ease of observation, methylene blue is used as the hydrogen acceptor after isolating the system from air, and the pyruvate generated during the fixed reaction completely converts all lactate into pyruvate. Today, we will introduce how to extract lactate dehydrogenase and coenzyme I using a benchtop centrifuge.

　　The materials, reagents, and equipment used for the centrifugal extraction of lactate dehydrogenase and its coenzyme include: (1) animal hind leg muscle; (2) 9 g/L NaCl solution; (3) activated carbon; (4) 10 g/L lithium lactate solution; (5) 5 g/L KCN solution; (6) mortar and pestle; (7) benchtop centrifuge TD5A-WS, etc.

　　Specific operating instructions:

　　1. Preparation of Coenzyme I (NAD⁺): Take 2 g of animal hind-leg muscle, place it in a boiling water bath, and boil for 4–5 minutes (to inhibit enzyme activity and prevent the destruction of Coenzyme I). Remove the muscle, allow it to cool, and chop it into small pieces. Add a small amount of 9 g/L NaCl solution and fine sand, then grind the mixture into a paste using a mortar and pestle. Next, add 8 ml of 9 g/L NaCl solution and mix thoroughly. Use a benchtop centrifuge to perform centrifugation and collect the supernatant. Store the supernatant in a refrigerator for later use.

　　2. Extraction of lactate dehydrogenase enzyme protein: Take 2 g of muscle from the hind leg of an animal, place it in a mortar, and add a small amount of 9 g/L NaCl solution along with fine sand. Grind thoroughly, then add 8 ml of 9 g/L NaCl solution and mix well. Filter the mixture through double-layer gauze. Add 0.5 g of activated carbon to the filtrate, shake vigorously for several minutes, and let it stand for half an hour, shaking occasionally. Then, centrifuge the mixture using a benchtop centrifuge to separate the supernatant, which can be stored for further use.

Conversion between centrifuge speed and centrifugal force

Centrifugal Separation Technique for Bacterial Cultures