大家都在搜
|
目前，HRP在免疫学实验ELISA及WB中应用非常广泛，其标记抗体的方法也比较多，一般有Glutaraldehyde法，NaIO4氧化法等，这里我们主要介绍的是在NaIO4氧化法基础上进行改进过的一种方法-------改良型NaIO4氧化法。 具体反应原理图如下：
主要标记反应步骤如下： 1. Dissolve the enzyme ( HRP）to be oxidized in water or 0.01 M sodium phosphate, 0.15 M NaCl, pH 7.2, at a concentration of 10–20 mg/ml and dissolve sodium periodate in water at a concentration of 0.088 M. 2. Immediately add 100 μL of the sodium periodate solution to each ml of the enzyme solution. This ratio of addition results in an 8 mM periodate concentration in the reaction mixture. Mix to dissolve. Protect from light. React in the dark for 15–20 minutes at room temperature. If HRP is the enzyme being oxidized, a color change will be apparent as the reaction proceeds—changing the brownish/gold color of concentrated HRP to green. 3. Purify the oxidized enzyme , we could dialyze and desalt in PBS 4. Dissolve the antibody to be conjugated at a concentration of 10 mg/ml in 0.1 M sodium bicarbonate, pH 9.6, at room temperature. The high-pH buffer will result in very efficient conjugation with the highest possible incorporation of enzyme molecules per antibody molecule. 5. Mix the antibody solution with the enzyme solution at a ratio of 1:1 (v/v). Typically, molar ratios of 4:1 to 15:1 ( enzyme: antibody ) give acceptable conjugates useful in a variety of ELISA techniques. 6. React for 2 hours at room temperature. 7. In a fume hood, add 10 μL of 5 M sodium cyanoborohydride per ml of reaction solution. All operations should be done with care in a fume hood. 8. React for 30 minutes at room temperature with gentle mixing (in a fume hood). 9. Block unreacted aldehyde sites by addition of 50 μL of 1 M ethanolamine, pH 9.6, per ml of conjugation solution. Approximately a 1 M ethanolamine solution may be prepared by addition of 300 μL ethanolamine to 5 ml of deionized water. Adjust the pH of the ethanolamine solution by addition of concentrated HCl, keeping the solution cool on ice.
10. React for 30 minutes at room temperature. 11. Purify the conjugate from excess reactants by dialysis against PBS or gel filtration 注：以上HRP标记抗体的方法也适用于标记链霉亲和素（Streptavidin），蛋白或者多肽等富含游离氨基的的物质，具体标记时所使用的比例需要重新计算。
|
