ATP硫酸化酶和亚硫酸还原酶的活力测定

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之前做了一个ATPase的 借鉴一下吧 Cell Membrane Preparation Cells were grown in DMEM/10% FBS with antibiotics plus fungizone until cells became 80-90% confluent and provided in the eppendorf tube labeled “Cells”. Cells were harvested with 500 μl cell suspension buffer (CBS) [buffer A (50 mM Tris, pH 8.0 and 0.5 mM dithiothreitol, DTT) and protease inhibitors (1 mM phenylmethylsulfonyl floride, 5 μg/ml leupeptin, 5 μg/ml aprotinin) plus phosphatase inhibitors (10 mM NaF and 1 mm Na3VO4)] in an eppendorf tube. Kept at -20oC until use. Thaw cells on ice. Cells were sheared by passing through a 25 gauge needle 25 times. The cell lysate was then spun at 1000 rpm for 10 minutes at 4oC. As much of the supernantant was transferred to a new eppendorf tube labeled “Supernantant 1” Resuspended the pellet in 200 μl CBS. Leave on ice for 10 minutes with occasional shaking. Respun the suspension at 1000 rpm for 20 minutes at 4oC. As much of the supernatant was then transferred to another new eppendorf tube labeled “Supernantant 2” Pellet was resuspended in 100 μl CBS. Sat on ice for 20 minutes with occasional shaking. Spun at 15000 rpm for 20 minutes at 4oC. The supernatant was then transferred to another new eppendorf tube labeled “Supernantant 3” Gathered “Supernantant 1” and “Supernantant 2”. This is the plasma membrane fraction. “Supernantant 3” is the post-plasma membrane fraction Protein Concentration Measurement (i) Protein Assay – Bradford Method RESULTS 1. From the table, construct a standard curve of amount of standard protein (μg) versus absorbance at 595 nm. Based on your standard curve, find out the protein concentration in μg/ml of your samples. Show both your standard curve and your calculations to your demonstrator for marking. If results of your samples are not within the reading range of the standard curve, repeat your test with appropriate amount of sample (e.g. by dilution or using a larger volume of sample)