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首页> 外文期刊>Journal of Neuroscience Methods >Generation of anti-peptide antibodies against serotonin 5-HT2A and 5-HT2C receptors.
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Generation of anti-peptide antibodies against serotonin 5-HT2A and 5-HT2C receptors.

机译：抗血清素5-HT2A和5-HT2C受体的抗肽抗体的产生。

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Anti-peptide antibodies were generated against several 13-17 amino acid regions of rat serotonin 5-HT2A and 5-HT2C receptors. Peptides containing terminal cysteine residues were conjugated to bovine serum albumin (BSA) and ovalbumin (OVA) with the cross-linking reagent sulfo-SMCC (sulfosuccinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate). Both the carrier protein and the number of peptide molecules per carrier molecule were changed during the immunization schedule. For the early immunizations, immunogens were BSA-peptides at ratios of 8-27 mol peptide per mol BSA. For the later boosts, immunogens were OVA-peptides at ratios of 1-2 mol peptide per mol OVA. The peptide constructs were used to immunize rabbits and chickens. Anti-peptide antibodies were purified from sera (rabbits) or egg yolks (hens) using peptide matrices. Cell lines expressing similar densities of rat 5-HT2A or 5-HT2C receptors were used to monitor the specificity of purified antibodies on immunoblots and in immunocytochemistry. A total of five out of the six rabbit antibodies were positive on immunoblots (three anti-5-HT2A and two anti-5-HT2C) and four were also positive in immunocytochemistry (three anti-5-HT2A and one anti-5-HT2C). None of the anti-peptide chicken antibodies were useful on immunoblots or in immunocytochemistry. Since there is a paucity of high affinity reagents selective for 5-HT2A or 5-HT2C receptors, these rabbit antibodies will be useful tools. The methods used to generate site-directed antibodies specific for 5-HT2A or 5-HT2C receptors should be applicable to other proteins.
机译：针对大鼠血清素5-HT2A和5-HT2C受体的几个13-17个氨基酸区域生成了抗肽抗体。用交联剂磺基-SMCC（磺基琥珀酰亚胺基4-（N-马来酰亚胺基甲基）环己烷-1-羧酸酯）将含有末端半胱氨酸残基的肽与牛血清白蛋白（BSA）和卵清蛋白（OVA）偶联。在免疫程序中，载体蛋白和每个载体分子的肽分子数量都发生了变化。对于早期免疫，免疫原是BSA-肽，其比例为每摩尔BSA 8-27摩尔肽。对于随后的增强，免疫原是1-2摩尔肽/摩尔OVA的比例的OVA肽。该肽构建体用于免疫兔和鸡。使用肽基质从血清（兔）或蛋黄（母鸡）中纯化抗肽抗体。表达相似密度的大鼠5-HT2A或5-HT2C受体的细胞系用于监测纯化抗体在免疫印迹和免疫细胞化学中的特异性。在六种兔抗体中，总共有五种对免疫印迹呈阳性（三种抗-5-HT2A和两种抗-5-HT2C），并且四种在免疫细胞化学中也呈阳性（三种抗-5-HT2A和一种抗-5-HT2C ）。抗肽鸡抗体中没有一种可用于免疫印迹或免疫细胞化学。由于缺乏对5-HT2A或5-HT2C受体具有选择性的高亲和力试剂，因此这些兔抗体将是有用的工具。用于产生特异于5-HT2A或5-HT2C受体的定点抗体的方法应适用于其他蛋白质。

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