Chloroplast complex V activity determination kit based on micro-method and method

A technology for activity measurement and complex, applied in the field of life sciences, can solve problems such as the method for determining the activity of chloroplast complex V that has not yet been discovered

Inactive Publication Date: 2019-04-09
SUZHOU COMIN BIOTECH
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Problems solved by technology

At present, there are few reports on the method for measuring the activity of mitochondrial complex V, but no effective method for detecting the activity of chloroplast complex V has been found.

Method used

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Embodiment Construction

[0092] The present invention will be described in detail below in conjunction with embodiments.

[0093] A chloroplast complex V activity assay kit based on the micro-method, characterized in that it includes the following reagents:

[0094] Reagent 1, liquid 100mL×1 bottle, mixed with 100mL, 50mM Tris-HCl (pH7.4), 10.96g sucrose, 380mg EGTA, stored at -20°C;

[0095] Reagent 2, powder × 1 tube, composed of 36.3mgATP, placed in a 2mL EP tube, stored at -20°C;

[0096] Reagent 3, liquid 8mL×1 bottle, composed of 8mL50mM Tris-HCl (pH7.2), 61mgMgCl 2 , 60mgKCl, 47mgNaCl mixed, stored at 4°C;

[0097] Reagent 4, powder x 1 bottle, composed of 0.8g TCA, placed in a 5mL reagent bottle, stored at 4°C;

[0098] Reagent 5, powder × 1 bottle, composed of 1g ASA, placed in a 10mL reagent bottle, stored at 4°C;

[0099] Reagent 6, powder x 1 bottle, composed of 0.25g ammonium molybdate, placed in a 10mL reagent bottle, stored at 4°C;

[0100] Reagent VII, liquid 10mL x 1 bottle, made...

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Abstract

The invention discloses a chloroplast complex V activity determination kit based on a micro-method and a method. The kit comprises a reagent 1 (Tris-HCl, sucrose, EGTA), a reagent 2 (ATP), a reagent 3(Tris-HCl, MgCl2, KCl, NaCl), a reagent 4 (TCA), reagent 5 (ASA), a reagent 6 (ammonium molybdate), and a reagent 7 (sulfuric acid, water). The method including extracting and detecting activity of chloroplast complex V is established for the first time, the principle is that complex V hydrolyzes ATP to produce ADP and Pi, and activity of complex V is determined by measuring the increasing rate of Pi; by reasonably arranging a contrast tube, the influence of impurities can be eliminated, and the detection specificity is very high; the method can effectively separate cytoplasmic protein, mitochondrial protein and chloroplast protein, and accurately measures the activity of complex V located in chloroplast; and the method has good repeatability, and the coefficient of variation is within 2%.

Description

technical field [0001] The invention belongs to the field of life sciences, and in particular relates to a micro-method-based chloroplast complex V activity assay kit and method. Background technique [0002] Chloroplast complex V also known as F 1 f 0 -ATP synthase, which uses the proton electrochemical gradient generated by the respiratory chain to catalyze ATP synthesis, and also hydrolyzes ATP in a reversible process. Complex V is the key enzyme for mitochondrial oxidative phosphorylation and chloroplast photophosphorylation to synthesize ATP. At present, there are few reports on the method for measuring the activity of complex V in mitochondria, but no effective method for detecting the activity of complex V in chloroplast has been found yet. Contents of the invention [0003] In order to overcome the above-mentioned defects in the prior art, the present invention aims to provide a chloroplast complex V activity assay kit and method based on the trace method, which...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/31G01N33/535G01N21/78
CPCG01N21/31G01N21/78G01N33/535
Inventor 吴华静王跃赵林川
Owner SUZHOU COMIN BIOTECH
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