Method for identification and purification of intracellular enolase

Abstract

The invention belongs to a method for identification and purification of intracellular enolase. The method comprises the method for identifying and purifying the intracellular enolase. The method comprises the following steps that: 0.5 g (wet weight) of collected bacteria or cells are suspended by using 0.5 ml of a PBS buffer; treatments of electrophoresis and gel stripping are performed; the resulting gel sheet is subjected to activity staining; a small amount purification treatment is performed by a dialysis method to obtain the enolase; and the like. With the present invention, the intracellular enolase can be intuitionally identified and purified, and the method has characteristics of simple operation, short experimental period, low price, economy and practicality.

Description

technical field [0001] The invention belongs to a method for identifying and purifying intracellular enolase. Background technique [0002] Enolase, also known as 2-phosphoglycerol hydrolase, is an intracellular enzyme necessary for glycolysis, which can catalyze the conversion of phosphoglycerate to phosphoenolpyruvate, and is also involved in the process of gluconeogenesis Catalyze the reverse reaction. The usual method for measuring the activity of the enzyme protein is to simulate part of the sugar metabolism reaction chain in vitro. The process is that enolase catalyzes 2-phosphoglycerate to form PEP (phosphoenolpyruvate), and PEP dephosphates to become pyruvate, which becomes lactic acid under the action of lactate dehydrogenase, and makes NADH + (reduced coenzyme I) into NAD (oxidized coenzyme I) and H + . Use a spectrophotometer with a wavelength of 340nm to measure the amount of NADH converted to NAD per unit time during the reaction, so as to determine the enol...

AI Technical Summary

Problems solved by technology

This method is a commonly used method now, but for the study of the characteristics of new bacterial enolases, this method can only be quantitatively detected, but cannot be visualized

For the intuitive identification method, the corresponding enzyme protein is often expressed by genetic engineering, which relies on the preparation of antibodies, and is identified by Western Blotting, ELISA and other methods. The experimental period is long, the price is very expensive, and it is not economical and practical.