使用说明：
1. 试剂盒的准备工作：
a.  配制250mM过氧化氢溶液。本试剂盒提供的过氧化氢浓度约为1M。由于过氧化氢不是非常稳定，使用
    前需自行测定过氧化氢的实际浓度。把浓度约为1M的过氧化氢用本试剂盒提供的过氧化氢酶检测缓
    冲液稀释100倍，使过氧化氢的浓度约为10mM。测定A₂₄₀。
    过氧化氢浓度(mM)=22.94 X A₂₄₀
    从而计算出本试剂盒提供的过氧化氢的实际浓度。然后再根据实际的过氧化氢浓度配制250mM过氧化
    氢溶液。
b.  配制5mM过氧化氢溶液。根据测定得到的实际过氧化氢浓度配制5mM过氧化氢溶液。
c.  配制显色工作液。在冰浴上溶解显色底物，适当分装后再使用，尽量避免反复冻融。其它试剂放置
    在冰浴上备用。取适当量的过氧化物酶，按照1:1000的比例用显色底物稀释，配制成显色工作液。
    例如取5μl过氧化物酶，加入5ml显色底物，混匀即得到5ml显色工作液。
2. 样品的准备：
    用适当的裂解液裂解细胞或组织(可以使用碧云天生产的Western及IP细胞裂解液(P0013)进行裂解。
    用本试剂盒提供的过氧化氢酶检测缓冲液稀释样品，裂解好的样品至少加入等体积的过氧化氢酶检
    测缓冲液进行稀释。具体的稀释倍数可以参考表1。
    表1

    说明：表中的数据仅供参考，实际测定值由于检测体系的不同等可能与参考值之间存在20-50%的误
    差。对于相同的样品，同样的实验体系，测得的A520值和表1相比可能会有较大差异。如果实验体系
    不同，例如测得蛋白浓度的方法不同，使用的检测波长不同等，测得的A520值和我们提供的参考数
    值相比可能会有更大的差异。对于表中没有列出的样品，可以参考表中的类似样品进行检测，然后
    再根据检测结果适当调整蛋白浓度和样品使用量，或反应时间。通常对于完全未知的样品，可以对
    样品进行1、10、20和50倍稀释，然后各取10μl，反应1分钟。样品稀释后的浓度，最好可以使反应
    体系中的过氧化氢在反应1-5分钟后减少30-50%左右，这时的检测结果更加精确。
a.  细胞样品的准备。可以用碧云天生产的Western及IP细胞裂解液(P0013)参考相应的说明裂解细胞样
    品。
b.  组织样品的准备。可以用碧云天生产的Western及IP细胞裂解液(P0013)参考相应的说明裂解组织样
    品。
c.  全血样品的准备。收集全血(whole blood)至一抗凝管内，颠倒混匀。取100微升全血冻融一次，用
    过氧化氢酶检测缓冲液稀释1000倍后进行后续检测。
d.  红细胞裂解液的准备。用抗凝管收集血液，颠倒混匀。取至少500微升全血4℃ 2500g离心5分钟。弃
    上清，沉淀用冰冷的生理盐水(0.9%氯化钠)洗涤3次。用约5倍细胞体积的冰冷的去离子水，例如
    Milli-Q纯水，重悬细胞沉淀，冰浴10分钟。使用前用过氧化氢酶检测缓冲液稀释400倍后进行后续
    检测。
3. 标准曲线测定：
a.  取0、12.5、25、50或75微升配制好的5mM过氧化氢溶液至1.5ml或0.5ml塑料离心管中，分别加入过
    氧化氢酶检测缓冲液至最后体积为100微升，混匀。
b.  各取4微升，加入到96孔板中的一个孔内。加入200μl显色工作液。25℃至少孵育15分钟后测定
    A₅₂₀，但孵育时间不宜超过45分钟。(注：本步骤可以和样品测定步骤中的最后一步同时进行。)
4. 样品测定：
    表2

a.  参考表2，取x微升(0-40微升)样品至1.5ml塑料离心管中，加入过氧化氢酶检测缓冲液至体积为40微
    升(即加入40-x微升过氧化氢酶检测缓冲液)，混匀。再加入10微升250mM过氧化氢溶液，用移液器迅
    速混匀。参考表1，25℃反应1-5分钟。
b.  加入450微升过氧化氢酶反应终止液，颠倒混匀或Vortex混匀以终止反应。需在终止反应后15分钟
    内完成下面的步骤c和步骤d。
c.  在一洁净的塑料离心管内加入40微升过氧化氢酶检测缓冲液，再加入10微升已终止并混匀的上述
    反应体系，混匀。
d.  从上一步骤的50微升体系中取10微升加入到96孔板中的一个孔内。加入200微升显色工作液。
e.  25℃至少孵育15分钟后测定A520，但孵育时间不宜超过45分钟。
5. 样品中过氧化氢酶酶活力的计算：
a.  计算出标准曲线。A₅₂₀＝k[过氧化氢微摩尔数]＋b，由标准曲线计算出k和b的值。
b.  计算出样品中残余的过氧化氢摩尔数。
    残余过氧化氢微摩尔数＝(A₅₂₀-b)/k
c.  过氧化氢酶酶活力单位的定义：1个酶活力单位(1 unit)在25℃，pH7.0的条件下，在1分钟内可以催
    化分解1微摩尔过氧化氢。
d1. 对于细胞或组织样品的过氧化氢酶活力计算：
    [样品过氧化氢酶酶活力]＝[消耗过氧化氢微摩尔数] X[稀释倍数]/([反应分钟数]X[样品体积] X
    [蛋白浓度])
    [样品过氧化氢酶酶活力]的单位为units/mg蛋白
    [消耗过氧化氢摩尔数]＝[空白对照残余过氧化氢微摩尔数]－[样品残余过氧化氢微摩尔数]
    [稀释倍数]＝250
    [反应分钟数]即为实际的反应分钟数
    [样品体积]为表2中的X微升，以毫升来表示即为X/1000毫升。
    [蛋白浓度]为取X微升样品时，样品中的蛋白浓度，单位为mg/ml。
d2. 对于血浆等液体样品的过氧化氢酶活力计算：
    [样品过氧化氢酶酶活力]＝[消耗过氧化氢微摩尔数] X[稀释倍数]/([反应分钟数]X[样品体积])
    [样品过氧化氢酶酶活力]的单位为units/ml样品

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