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组织细胞总蛋白抽提试剂盒 Histocyte Total Protein Extraction Kit P1250厂家直销,提供OEM定制服务!
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    P1250-50
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描述:从组织细胞中提取总蛋白是Western Blot的关键步骤。实体软组织如脑脊髓富含磷脂,神经血管含大量结缔组织,而脂肪则含有大量油脂,常规的方法难以有效从这些组织中提取蛋白。本试剂盒为组织或培养细胞总蛋白提取提供完整的解决方案。用裂解-结合缓冲液匀浆裂解实体组织,或直接用裂解-结合缓冲液重悬培养细胞,然后加入抽提试剂去除非蛋白成分,离心、干燥后即可得到总蛋白。蛋白沉淀溶解后用常规方法进行蛋白定量。提取过程可在30-60分钟内完成,可在1.5mL离心管微量提取也可大规模制备,极为简便高效。

规格:

50 100次

储存:

室温或4ºC 24个月有效

操作步骤:

固体组织蛋白提取

1. 10-100mg固体组织剪碎后加入0.5-1mL裂解液,放入玻璃匀浆器内上下手动匀浆15次;或用高速机械匀浆器12,000rpm破碎组织。少量小的未破碎组织不影响后续抽提。注意:肝肾脾组织蛋白含量高,提取时应加较少量的组织,一般不超过50mg,否则形成的蛋白膜厚、致密且难溶。

2. 仅取0.5mL组织匀浆液转移到1.5mL离心管,弃去剩余的组织匀浆液。每0.5mL组织匀浆液加入2倍体积的(1mL)抽提试剂充分混匀。室温或4ºC 静置10分钟,偶尔晃动。注意:(1)如组织量<10mg,在下一步离心时形成的蛋白膜易碎,静置30min有助蛋白膜形成。(2)提取脂肪组织时应4ºC 静置40min 以上以充分去除油脂。(3)0.5mL组织匀浆液获得的蛋白足以进行几十次Western Blot。保证每0.5mL 组织匀浆液加入2 倍体积的(1 mL)抽提试剂是成功的关键。

3. 10,000×g4ºC 离心10分钟,溶液分为上下两相,两相中间为蛋白膜。吸除上层液体;随后用吸头或针头轻轻拨开蛋白膜,吸除下层液体。蛋白膜将附着于离心管壁。注意:(1)离心速度过高将使蛋白膜过于坚固,难以溶解。(2)如果不 能分相,可再加入50μl 蒸馏水混匀离心.(3)如果初始组织量较少(<10mg),离心后难以得到完整的蛋白膜,此时可吸去上下层液体,加入1mL 纯乙醇混匀,10,000 ×g 4ºC 离心3 分钟。弃所有液体,蛋白沉淀在管底。

4. 敞开管口,室温空气干燥沉淀。未溶解的蛋白沉淀不含盐、去垢剂、和还原剂成分,可4ºC或-20ºC一年以上。

培养细胞蛋白提取 (参见固体组织蛋白提取程序中的斜体字注解)

1. 消化洗涤并800g离心收集细胞。每5-10×106细胞加入0.5mL裂解液,振荡重悬,4ºC净置2分钟。

2. 按比例每0.5mL裂解液加入1mL抽提试剂,振荡混匀。4ºC 静置10min

3. 10,000g4ºC 离心10分钟,溶液分为两相,两相中间为蛋白膜。小心吸除上层相和大部分下层相,保留两相中间的蛋白絮状物。如果不能分相,可再加入50μl蒸馏水混匀离心。

4. 加入1mL纯乙醇洗涤沉淀。10,000g4ºC 离心3分钟,蛋白沉淀在管底。

5. 去除管中所有液体,敞开管口,室温空气干燥蛋白沉淀。未溶解的蛋白沉淀不含盐、去垢剂、和还原剂成分,可4ºC或-20ºC一年以上。

说明

1. 按照上述提取和溶解过程,不加蛋白酶抑制剂,而蛋白不会有明显降解,用户不必(但可以)加入蛋白酶抑制剂。

2. 蛋白定量。注意使SDS浓度稀释到不影响蛋白定量的水平,或选择不受SDS影响的BCA定量方法.BCA<5%SDS浓度,Bradford<0.125%SDS浓度。

3. 按比例可进行大规模的(多至1g)组织蛋白提取。

4. 提取试剂对眼睛和呼吸系统有一定刺激性,皮肤不慎接触可用水清洗。



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