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上海研卉生物科技有限公司
200T
样品实验方案
简要概述
1.准备G6PD工作溶液(50 µL)
2.添加G6PD标准品或测试样品(50 µL)
3.在室温下孵育30分钟-2小时
4.监测在A575nm / A605nm处的吸光度比增加
溶液制备
1.储备溶液
所有未使用的储备溶液应分为一次性使用的等分试样,并在制备后储存在-20°C下。 避免重复冻融循环。
1.1 NADP储备溶液(100X):
在NADP小瓶(组分C)中加入100 µL H2O,制成100X NADP储备溶液。
1.2 G6PD标准溶液(100 U / mL):
将100 µL H2O或1X PBS缓冲液加到G6PD Standard(组分D)的小瓶中,制成100 U / mL G6PD标准溶液。
2.标准溶液
G6PD标准
将10 µL 100 U / mL G6PD标准溶液添加到990 µL 1x PBS缓冲液中,以生成1000 mU / mL G6PD标准溶液。 取1000 mU / mL G6PD标准溶液,并在1x PBS缓冲液中进行1:3系列稀释,以得到系列稀释的G6PD标准液(G6PD7-G6PD1)。 注意:稀释的G6PD标准溶液不稳定,应在4小时内使用。
3.工作溶液
3.1 将5 mL测定缓冲液(组分B)加入一瓶酶探针(组分A)中,并充分混合。
3.2 将50 µL 100X NADP储备溶液添加到组分A + B的瓶子中,并充分混合以制成G6PD工作溶液。 注意:此G6PD工作溶液足以用于一个96孔板。 它在室温下不稳定,应在2小时内及时使用。 避免暴露在光线下。
样品操作及实验分析
表1. G6PD标准品和测试样品在白色透明底部96孔微孔板中的布局。 G6PD = D-葡萄糖-6-磷酸脱氢酶标准品(G6PD1-G6PD7,0.4至300 mU / mL),BL =空白对照,TS =测试样品。
BL | BL | TS | TS |
G6PD1 | G6PD1 | ... | ... |
G6PD2 | G6PD2 | ... | ... |
G6PD3 | G6PD3 | ||
G6PD4 | G6PD4 | ||
G6PD5 | G6PD5 | ||
G6PD6 | G6PD6 | ||
G6PD7 | G6PD7 |
表2.每个孔的试剂组成
孔 | 容积 | 试剂 |
G6PD1-G6PD7 | 50ul | 连续稀释液(0.4至300 mU / mL) |
BL | 50ul | 稀释缓冲液 |
TS | 50ul | 测试样品 |
1.根据表1和表2提供的布局,准备G6PD标准品(G6PD),空白对照(BL)和测试样品(TS)。对于384孔板,每孔使用25 µL试剂代替50 µL。
2.向G6PD标准品,空白对照和测试样品的每个孔中加入50 µL G6PD工作溶液,以使G6PD测定总体积为100 µL /孔。 对于384孔板,将25 µL G6PD工作溶液添加到每个孔中,总体积为50 µL /孔。
3.避光保存,室温下孵育反应30分钟至2小时。
4.使用酶标仪在A575nm / A605nm处监测吸光度的增加。
参考文献
The microsomal enzyme 17beta-hydroxysteroid dehydrogenase 3 faces the cytoplasm and uses NADPH generated by glucose-6-phosphate dehydrogenase
Authors: Legeza B, Balazs Z, Nashev LG, Odermatt A.
Journal: Endocrinology (2013): 205
A novel cytofluorometric assay for the detection and quantification of glucose-6-phosphate dehydrogenase deficiency
Authors: Shah SS, Diakite SA, Traore K, Diakite M, Kwiatkowski DP, Rockett KA, Wellems TE, Fairhurst RM.
Journal: Sci Rep (2012): 299
Effects of glucose-6-phosphate dehydrogenase deficiency on the metabolic and cardiac responses to obesogenic or high-fructose diets
Authors: Hecker PA, Mapanga RF, Kimar CP, Ribeiro RF, Jr., Brown BH, O'Connell KA, Cox JW, Shekar KC, Asemu G, Essop MF, Stanley WC.
Journal: Am J Physiol Endocrinol Metab (2012): E959
Effects of laparoscopic Roux-en-Y gastric bypass on glucose-6 phosphate dehydrogenase activity in obese type 2 diabetics
Authors: Schneider AM, Rawat D, Weinstein LS, Gupte SA, Richards WO.
Journal: Surg Endosc (2012): 823
High prevalence of hemoglobin disorders and glucose-6-phosphate dehydrogenase (G6PD) deficiency in the Republic of Guinea (West Africa)
Authors: Millimono TS, Loua KM, Rath SL, Relvas L, Bento C, Diakite M, Jarvis M, Daries N, Ribeiro LM, Manco L, Kaeda JS.
Journal: Hemoglobin (2012): 25
Molecular characterization of glucose-6-phosphate dehydrogenase deficient variants in Baghdad city - Iraq
Authors: Al-Musawi BM, Al-Allawi N, Abdul-Majeed BA, Eissa AA, Jubrael JM, Hamamy H.
Journal: BMC Blood Disord (2012): 4
Alternative targeting of Arabidopsis plastidic glucose-6-phosphate dehydrogenase G6PD1 involves cysteine-dependent interaction with G6PD4 in the cytosol
Authors: Meyer T, Holscher C, Schwoppe C, von Schaewen A.
Journal: Plant J (2011): 745
Frequency of glucose-6-phosphate dehydrogenase deficiency in malaria patients from six African countries enrolled in two randomized anti-malarial clinical trials
Authors: Carter N, Pamba A, Duparc S, Waitumbi JN.
Journal: Malar J (2011): 241
Glucose-6-phosphate dehydrogenase status and severity of malarial anaemia in Nigerian children
Authors: Orimadegun AE, Sodeinde O.
Journal: J Infect Dev Ctries (2011): 792
Implementation and analysis of a pilot in-hospital newborn screening program for glucose-6-phosphate dehydrogenase deficiency in the United States
Authors: Nock ML, Johnson EM, Krugman RR, Di Fiore JM, Fitzgerald S, Sandhaus LM, Walsh MC.
Journal: J Perinatol (2011): 112
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