Schmidt M L, Trojanowski J Q
Anal Biochem. 1986 Jun;155(2):371-5. doi: 10.1016/0003-2697(86)90448-3.
We developed procedures for the restoration of peroxidatic activity in native horseradish peroxidase (HRP) and HRP conjugated to wheat germ agglutinin (WGA-HRP) following electrophoresis in SDS-polyacrylamide gels (SDS-PAGE). After extraction of SDS with isopropanol from gels containing HRP and WGA-HRP, the peroxidatic activity in these probes could be demonstrated by tetramethylbenzidine (TMB) chemistry. This procedure also showed HRP enzyme activity in electrophoresed tissue homogenates containing HRP. Both free HRP as well as WGA-HRP preparations contain several molecular weight species that display peroxidatic activity. These findings are important for cell biological studies utilizing these substances as molecular probes. The procedures described here should be useful for the analysis of the enzymatically active molecular forms of these frequently used markers in vitro and in vivo.
我们开发了在十二烷基硫酸钠-聚丙烯酰胺凝胶（SDS-PAGE）电泳后恢复天然辣根过氧化物酶（HRP）和与麦胚凝集素偶联的HRP（WGA-HRP）中过氧化物酶活性的方法。在用异丙醇从含有HRP和WGA-HRP的凝胶中提取SDS后，这些探针中的过氧化物酶活性可以通过四甲基联苯胺（TMB）化学方法来证明。该方法还显示了在含有HRP的电泳组织匀浆中的HRP酶活性。游离HRP以及WGA-HRP制剂都包含几种具有过氧化物酶活性的分子量种类。这些发现对于利用这些物质作为分子探针的细胞生物学研究很重要。这里描述的方法对于分析这些常用标记物在体外和体内的酶活性分子形式应该是有用的。
