小鼠内脂素/内脏脂肪素（visfatin）ELISA试剂盒

 小鼠内脂素/内脏脂肪素（visfatin）ELISA试剂盒含税含运费　, 
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    产品规格：96T/48T 
    96T指可以做94个标本，2个标准对照 
    48T指可以做47个标本，1个标准对照 
    96T-84个样本 
    48T-42个样本 
    操作步骤 
    1.标准品的稀释：本试剂盒提供原倍标准品一支，用户可按照下列图表在小试管中进行稀释。 
    3600 pg/ml5号标准品150μl的原倍标准品加入150μl标准品稀释液 
    1800pg/ml4号标准品150μl的5号标准品加入150μl标准品稀释液 
    900pg/ml3号标准品150μl的4号标准品加入150μl标准品稀释液 
    450 pg/ml2号标准品150μl的3号标准品加入150μl标准品稀释液 
    225pg/ml1号标准品150μl的2号标准品加入150μl标准品稀释液 
     
     
    2.加样：分别设空白孔（空白对照孔不加样品及酶标试剂，其余各步操作相同）、标准孔、待测样品孔。在酶标包被板上标准品准确加样50μl，待测样品孔中先加样品稀释液40μl，然后再加待测样品10μl（样品zui终稀释度为5倍）。加样将样品加于酶标板孔底部，尽量不触及孔壁，轻轻晃动混匀。 
    3.温育：用封板膜封板后置37℃温育30分钟。 
    4.配液：将20倍浓缩洗涤液用蒸馏水20倍稀释后备用 
    5.洗涤：小心揭掉封板膜，弃去液体，甩干，每孔加满洗涤液，静置30秒后弃去，如此重复5次，拍干。 
    6.加酶：每孔加入酶标试剂50μl，空白孔除外。 
    7.温育：操作同3。 
    8.洗涤：操作同5。 
    9.显色：每孔先加入显色剂A50μl，再加入显色剂B50μl，轻轻震荡混匀，37℃避光显色15分钟. 
    10.终止：每孔加终止液50μl，终止反应（此时蓝色立转黄色）。 
    11.测定：以空白空调零，450nm波长依序测量各孔的吸光度（OD值）。 测定应在加终止液后15分钟以内进行。 
    Assay procedure 
    1.Dilute and add sample:Dilute Original density Standard as follow table: 
    800 nmol/L5 Standard150μl Original density Standard+150μl Standard diluent 
    400 nmol/L4 Standard150μl 5 Standard+150μl Standard diluent 
    200 nmol/L3 Standard150μl 4 Standard+150μl Standard diluent 
    100 nmol/L2 Standard150μl 3 Standard +150μl Standard diluent 
    50 nmol/L1 Standard150μl 2 Standard +150μl Standard diluent 
    2.add sample：Set blank wells separay （blank comparison wells don’t add sample and HRP-Conjugate reagent, other each step operation is same）. testing sample well. add Sample dilution 40μl to testing sample well, then add testing sample 10μl （sample final dilution is 5-fold）, add sample to wells , don’t touch the well wall as far as possible, and Gently mix. 
    3.Incubate: After closing plate with Closure plate membrane ,incubate for 30 min at 37℃. 
    4.Configurate liquid: 30-fold wash solution diluted 30-fold （or 20-fold） with distilled water and reserve. 
    5.washing：Uncover Closure plate membrane, discard Liquid, dry by swing, add washing buffer to every well, still for 30s then drain, repeat 5 times, dry by pat. 
    6.add enzyme：Add HRP-Conjugate reagent 50μl to each well, except blank well. 
    7.incubate：Operation with 3. 
    8.washing：Operation with 5. 
    9.color：Add Chromogen Solution A 50ul and Chromogen Solution B to each well, evade the light preservation for 15 min at 37℃ 
    10.Stop the reaction：Add Stop Solution50μl to each well, Stop the reaction（the blue color change to yellow color）. 
    11.assay：take blank well as zero , Read absorbance at 450nm after Adding Stop Solution and within 15min. 
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