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保存条件 :4°保存
保质期 : 12个月有效
英文名 :Mem Preparation Kit
库存 :大量
供应商 :北京普利莱
CAS号 ://
规格 :50次/100次
细胞膜蛋白提取试剂盒(Mem Preparation Kit) P1203描述:本试剂盒能够从哺乳动物新鲜或冻存的组织块、贴壁或悬浮细胞中制备细胞膜与胞内质膜组分。其独特的试剂成分与优化的制备方案使胞膜制备过程简单易行,无需特殊设备和超速离心,可在1小时内完成。应用本试剂盒制备的胞膜是细胞膜和细胞器膜如线粒体、内质网及质膜的混合物。制备得到的产物纯度可胜任后续的免疫沉淀、蛋白印迹、2-D gel、酶活性检测和受体分析等实验。
组成: (以下是50次规格用量)
CER (Cytosol Extraction Reagent) 25ml
MER (Membran Extraction Reagent) 2.5ml
Suspension Buffer 10ml
储存:各组分4 oC 有效期12个月
操作步骤:
- 样本预处理:
- 贴壁细胞:用PBS缓冲液冲洗细胞平皿,用胰蛋白酶消化细胞。800g离心5-10分钟,弃上清,用PBS缓冲液重悬洗涤细胞并再次离心收集细胞。
- 悬浮细胞:800g离心5-10分钟,弃上清。用PBS缓冲液重悬洗涤细胞并再次离心收集细胞。
- 组织细胞匀浆裂解
- 细胞匀浆裂解
注意:此破碎细胞步骤为关键环节。要使用1-3ml小容积玻璃匀浆器,须选用间隙严密的研杵,其特征是将研杵插入匀浆器套管后,可提起研杵而套管不会脱落。有效研磨是上下推拉而不是旋转。破碎效果与细胞类型有关,可在相差显微镜下检查,未裂解细胞应小于5%。
- 组织块匀浆裂解:
注意:与培养细胞特别是贴壁细胞相比,组织块中的细胞在匀浆时较易破碎,因而并非必须选择间隙严密的研杵。如果研杵与套管过于严密,会使组织匀浆困难,可选用研杵与套管稍松的匀浆器,破碎效果与组织细胞类型有关,可在相差显微镜下检查,未裂解细胞应少于5%。
- 粗提物制备:
- 取约500µ裂解物,转移到新的离心管中,800g ,4oC离心5分钟。上清为胞膜-胞浆混合物。
- 胞膜胞浆制备:
说明:
- Suspension Buffer 不含去垢剂,重悬于Suspension Buffer 中的胞膜或胞核组分呈不溶解状态是正常的,用户可用自备溶液重悬胞膜或胞核成分。如果进行蛋白印迹、2-D get等实验,用户可用SDS上样缓冲直接裂解细胞膜或细胞核成分。对于进行免疫共沉淀实验来说,可用RIPA裂解液(C1053)重悬胞膜。
- 试剂盒各组分中未添加蛋白酶抑制剂,可自行选择添加。
以下仅列举数篇使用普利莱P1203试剂盒发表的文章供参考。
- Wu S, Yue Y, Tian H, et al. Tramiprosate protects neurons against ischemic stroke by disrupting the interaction between PSD95 and nNOS[J]. Neuropharmacology, 2014, 83: 107-117.
- Xu S, Wang P, Wei K, et al. Cytoprotection of perfluorocarbon on pmvecs in vitro[J]. Inflammation, 2013, 36(2): 512-520.
- Yao X, Chen H, Li Y. Protective effect of bicyclol on liver injury induced by hepatic warm ischemia/reperfusion in rats[J]. Hepatology Research, 2009, 39(8): 833-842.
- Yan S, Zhou C, Zhang W, et al. β-Catenin/TCF pathway upregulates STAT3 expression in human esophageal squamous cell carcinoma[J]. Cancer letters, 2008, 271(1): 85-97.
- Shi L L, Yang W N, Chen X L, et al. The protective effects of tanshinone IIA on neurotoxicity induced by β-amyloid protein through calpain and the p35/Cdk5 pathway in primary cortical neurons[J]. Neurochemistry international, 2012, 61(2): 227-235.
- Yang X Y, Zhao N, Liu Y Y, et al. Inhibition of NADPH oxidase mediates protective effect of cardiotonic pills against rat heart ischemia/reperfusion injury[J]. Evidence-Based Complementary and Alternative Medicine, 2013, 2013.
- Song X Y, Xu S, Hu J F, et al. Piperine prevents cholesterol gallstones formation in mice[J]. European journal of pharmacology, 2015, 751: 112-117.
- ZHAO H A I P, Feng J, Sun K, et al. Caffeic Acid Inhibits Acute Hyperhomocysteinemia‐Induced Leukocyte Rolling and Adhesion in Mouse Cerebral Venules[J]. Microcirculation, 2012, 19(3): 233-244.
- Hao H F, Liu L M, Liu Y Y, et al. Inhibitory effect of rhynchophylline on contraction of cerebral arterioles to endothelin 1: Role of rho kinase[J]. Journal of ethnopharmacology, 2014, 155(1): 147-153.
- Li A Q, Zhao L, Zhou T F, et al. Exendin-4 promotes endothelial barrier enhancement via PKA-and Epac1-dependent Rac1 activation[J]. American Journal of Physiology-Cell Physiology, 2015, 308(2): C164-C175.
- Zhao L, Li A Q, Zhou T F, et al. Exendin-4 alleviates angiotensin II-induced senescence in vascular smooth muscle cells by inhibiting Rac1 activation via a cAMP/PKA-dependent pathway[J]. American Journal of Physiology-Cell Physiology, 2014, 307(12): C1130-C1141.
- Tai W, Ye X, Bao X, et al. Inhibition of Src tyrosine kinase activity by squamosamide derivative FLZ attenuates neuroinflammation in both in vivo and in vitro Parkinson's disease models[J]. Neuropharmacology, 2013, 75: 201-212.
- Wang Q, Lin Y, Zhang W, et al. Lead induces dysregulation of iron regulatory protein 1 via the extracellular signal-regulated kinase pathway in human vascular endothelial cells[J]. Brain research, 2012, 1455: 19-27.
- Miao Y, Dong L D, Chen J, et al. Involvement of calpain/p35-p25/Cdk5/NMDAR signaling pathway in glutamate-induced neurotoxicity in cultured rat retinal neurons[J]. PloS one, 2012, 7(8): e42318.
- Liu X D, Yang J J, Fang D, et al. Functional Upregulation of Nav1. 8 Sodium Channels on the Membrane of Dorsal Root Ganglia Neurons Contributes to the Development of Cancer-Induced Bone Pain[J]. PloS one, 2014, 9(12): e114623.
- Liu M, Yang H, Fang D, et al. Upregulation of P2X3 receptors by neuronal calcium sensor protein VILIP-1 in dorsal root ganglions contributes to the bone cancer pain in rats[J]. Pain, 2013, 154(9): 1551-1568.
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