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[求助]
ADH酶活测定 已有1人参与
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| 求ADH酶活测定的具体流程，从试剂配制到结果结算，或者直接提供参考书也行~~~ |
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感谢参与，应助指数 +1
FLLY-1219: 金币+8, ★有帮助 2015-08-17 07:48:54
感谢参与，应助指数 +1
FLLY-1219: 金币+8, ★有帮助 2015-08-17 07:48:54
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没具体做过，帮你看了一下，首先应该有试剂盒可以用，其次，自己做的话，可以参考下文 Alcohol Dehydrogenase and Pyruvate Decarboxylase Activity in Leaves and Roots of Eastern Cottonwood (Populus deltoides Bartr.) and Soybean (Glycine max L.) THOMAS W. KIMMERER Plant Physiol. (1987) 84, 1210-1213 Enzyme Extractions. In preliminary experiments, the enzyme isolation procedure was optimized for each plant species and organ (leaf or root), with the use of large amounts of cofactors, reductants and phenolic binding agents necessary for enzyme isolation from woody plants (see Ref. 5 for a discussion). Cottonwood leaves were ground in a mortar and pestle at 4°C in grinding buffer (100 mM HEPES, 2 mm MgC12, 1 mm NAD, 2 mm TPP, 1 g PolyClar AT [GAF Corporation, New York], 100 mm EtSH [pH 7.55 at 4C]) at a ratio of 10 ml buffer/g fresh weight plant tissue, and the homogenate was filtered through Miracloth (Calbiochem). TPP and MgC12 were added to improve recovery of PDC. Their presence had no effect on recovery of ADH. The filtrate was centrifuged at l0,000g for 20 min at 4°C. The supernatant was then passed through a Sephadex G-25 column (Pharmacia) to remove low mol wt substances, with buffer exchange into holding buffer (100 mm HEPES, 2 mM MgCl2, 1 mM DTT, 2 mm TPP [pH 7.55] at 4°C), and the protein fraction was collected. The preparation was kept at 1°C and assayed within 2 h. For cottonwood roots, the same procedure was used, except that the EtSH concentration in the grinding buffer was reduced to 20 mm, and the PolyClar AT was reduced to 500 mg. For isolation of ADH from soybean leaves and roots, the EtSH concentration was reduced to 10 mm, and the pH was reduced to 7.4. PolyClar AT was omitted from the grinding medium for soybean roots. Enzyme Assays. Assay conditions were optimized for each plant species and tissue type. The preparations were assayed for ADH by following the oxidation of NADH at 340 nm. The reaction buffer for cottonwood extracts consisted of 100 mM Mes, 5 mM MgC12, 1 mm DTT (pH 6.25 at 25°C). To 2 ml reaction buffer, 250 ,ug NADH was added, followed by 100 ,1 protein extract. The A340 was followed for 2 min for determination of NADH oxidase activity, after which the reaction was started by addition of 100 ,A 70% acetaldehyde. The A340 was measured for 2 to 5 min. Results were calculated as ,/mol NADH oxidized min-' g-' dry weight after correction for NADH oxidase activity. For soybean roots, the optimum pH was 6.5 at 25°C. For soybean leaves, optimization was not possible due to lack of detectable ADH activity. Cottonwood leaf preparations were also analyzed with a variety ofaldehydes as substrate, including trans- 2-hexenal, propionaldehyde, and formaldehyde. Trans-2-hexenal was emulsified in the buffer by sonication just prior to addition of protein. PDC activity was measured by coupling pyruvate decarboxylation to NADH oxidation by ADH. The reaction buffer consisted of 100 mM Mes, 5 mM MgCl2, 1 mM DTT, 2 mm TPP (pH 6.1 at 25°C). NADH (250 Mg) was added to 2 ml of reaction buffer, followed by 100 ul protein extract and 30 units of yeast ADH. NADH oxidase activity was determined by measuring A340 for 2 min prior to the addition of 3.7 ,g pyruvic acid. The reaction was then followed for 10 min. Protein concentrations were measured by the method of Bradford (1), using purified spinach RuBPCase (Sigma) as standard, and BioRad Bradford reagent (BioRad). The fresh weight/dry weight conversion was determined on a subsample of each organ harvested for enzyme extraction. Sephadex was obtained from Pharmacia Fine Chemicals. All other reagents and enzymes were obtained from Sigma. Pyruvate was passed through Sephadex LH-20 to remove polymerized material just before use. |
2楼2015-08-15 17:28:55
3楼2015-08-15 17:40:40
