S7150
OxyBlot 蛋白质氧化检测试剂盒
The OxyBlot Protein Oxidation Detection Kit provides the reagents to perform the immunoblot detection of carbonyl groups introduced into proteins by oxidative reactions with ozone or oxides of nitrogen or by metal catalyzed oxidation.
别名:
OxyBlot检测, 蛋白质氧化试剂盒
一般描述
应用
1.用冰冷的PBS将培养皿或烧瓶中的贴壁细胞洗涤两次，并排出PBS。用PBS洗涤非贴壁细胞，在台式离心机中800~1000 rpm离心5分钟，使细胞团块。
2.向细胞中加入冰冷的RIPA缓冲液（1 ml/107个细胞/100 mm培养皿/150 cm2烧瓶；0.5 ml/5×106个细胞/60 mm培养皿/75 cm2烧瓶）。
3.用橡胶淀帚或已在冰冷蒸馏水中冷却的塑料细胞刮刀将贴壁细胞从培养皿或烧瓶中刮下。将细胞悬液转移至离心管中。在冷藏室的摇杆或定轨摇床上轻轻摇动混悬液15分钟以裂解细胞。
4.将裂解液在预冷离心机中以14,000 x g离心15分钟。立即将上清转移到新的离心管中，弃去沉淀。
5..在测定蛋白浓度之前将细胞裂解液以至少1：10稀释，因为裂解缓冲液中的洗涤剂对考马斯试剂有干扰。在该步骤中，可将样品分成等分试样，并在-20¡C下保存长达一个月。
TIP：当与大量非贴壁细胞一起工作时，细胞可能没有足够快地冷却以维持所研究蛋白质的活性。在这种情况下，将细胞悬液倒入等质量的2 x PBS和冰的混合物中，然后通过离心收集细胞并如上所述进行裂解。
RIPA裂解缓冲液：
目录编号20-188
http://www.millipore.com/catalogue/item/20-188
还原剂对防止氧化非常重要：
可以使用多种方案制备蛋白质裂解物。大多数标准缓冲液如RIPA和Triton也适用。建议在裂解缓冲液中加入还原剂，以防止细胞裂解后可能发生的蛋白质氧化。含有1-2% 2-巯基乙醇或50 mM DTT的裂解缓冲液足以抑制这种氧化，但对OxyBlot 方案中的衍生化反应没有不利影响。纯化的蛋白质样品也适用于使用OxyBlot 试剂盒进行分析。
有关蛋白提取的一般资料，请查此链接。
http://www.millipore.com/immunodetection/id3/proteinextraction
制备说明
法律信息
免责声明
警示用语：
Warning
危险声明
危险分类
Met. Corr. 1 - Skin Sens. 1
储存分类代码
8A - Combustible corrosive hazardous materials
相关内容
OxyBlot Protein Oxidation Detection Kit
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