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- 文献和实验
- 技术资料
- 库存:
1000
- CAS号:
-
- 保质期:
1年
- 供应商:
兰博利德
- 保存条件:
-20保存
- 规格:
100ml
保存条件: -20℃。保质期1年
产品简介:
RIPA裂解液(RIPA Lysis Buffer)是一种传统的细胞组织快速裂解液。RIPA裂解液裂解得到的蛋白样品可以用于常规的Western、IP等。
RIPA裂解液(强)的主要成分为50mM Tris(pH 7.4),150mM NaCl,1%的triton ,1% sodium deoxycholate,0.1% SDS,以及sodium orthovanadate,EDTA,leupeptin等多种抑制剂。可以有效抑制蛋白降解。
产品中包含一支1mL的PMSF(浓度100mM),无需自备。
注意事项:
1、 为取得最佳的使用效果,尽量避免过多的反复冻融。可以适当分装后使用。
2、裂解样品的所有步骤都需在冰上或4℃进行。
使用说明:
对于培养细胞样品:
1. 融解RIPA裂解液,混匀。取适当量的裂解液,在使用前数分钟内加入PMSF,使PMSF的最终浓度为1mM。
2. 对于贴壁细胞:去除培养液,用PBS、生理盐水或无血清培养液洗一遍(如果血清中的蛋白没有干扰,可以不洗)。按照6孔板每孔加入150-250微升裂解液的比例加入裂解液。用枪吹打数下,使裂解液和细胞充分接触。通常裂解液接触细胞1-2秒后,细胞就会被裂解。
对于悬浮细胞:离心收集细胞,用手指把细胞用力弹散。按照6孔板每孔细胞加入150-250微升裂解液的比例加入裂解液。再用手指轻弹以充分裂解细胞。充分裂解后应没有明显的细胞沉淀。如果细胞量较多,必需分装成50-100万细胞/管,然后再裂解。
3. 充分裂解后,10000-14000g离心3-5分钟,取上清,即可进行后续的PAGE、Western和免疫沉淀等操作。
裂解液用量说明:通常6孔板每孔细胞加入150微升裂解液已经足够,但如果细胞密度非常高可以适当加大裂解液的用量到200微升或250微升。
对于组织样品:
1. 把组织剪切成细小的碎片。
2. 融解RIPA裂解液,混匀。取适当量的裂解液,在使用前数分钟内加入PMSF,使PMSF的最终浓度为1mM。
3. 按照每20毫克组织加入150-250微升裂解液的比例加入裂解液。(如果裂解不充分可以适当添加更多的裂解液,如果需要高
浓度的蛋白样品,可以适当减少裂解液的用量。)
4. 用玻璃匀浆器匀浆,直至充分裂解。
5. 充分裂解后,10000-14000g离心3-5分钟,取上清,即可进行后续的PAGE、Western和免疫沉淀等操作。
6. 如果组织样品本身非常细小,可以适当剪切后直接加入裂解液裂解,通过强烈vortex使样品裂解充分。然后同样离心取上清,用于后续实验。直接裂解的优点是比较方便,不必使用匀浆器,缺点是不如使用匀浆器那样裂解得比较充分。
注:RIPA裂解液的裂解产物中经常会出现一小团透明胶状物,属正常现象。该透明胶状物为含有基因组DNA等的复合物。在不检测和基因组DNA结合特别紧密的蛋白的情况下,可以直接离心取上清用于后续实验;如果需要检测和基因组结合特别紧密的蛋白,则可以通过超声处理打碎打散该透明胶状物,随后离心取上清用于后续实验。如果检测一些常见的转录因子,例如NF-kappaB、p53等时,通常不必进行超声处理,就可以检测到这些转录因子。
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1ml RIPA buffer contains 35μl proteinase inhibitor(sigma cat no P8340) and 10μl phosphatase inhibitorRIPA buffer 最终浓度1M Tris-HCl pH7.5) 5 ml 50mMNaCl 0.87g 0.15 MNa-deoxycholale 0.1g (1%)0.5M EDTA(pH8.0) 0.8mlNaF 41 mgNonidet P40 1 ml (1%)Aprotinin
2,左条带为使用普通强裂解液提取的蛋白样品,右条带为柱式法提取的蛋白样品,可见使用柱式法法提取的样品胶孔中可明显改善样品滞留、拖带的情况发生。 图 2:同种样品分别使用裂解液法和柱式法进行 WB 样品制备,SDS-PAGE 电泳后考染图 图片来源:英文特 WB 蛋白样品制备过程中出现样品粘稠的情况并不代表样品提取失败,而是样品裂解较为充分的表现,但是如果放任不管则会引发一系列连锁效应,最终导致 WB 实验失败。选择合适的方法进行处理。成功获得高质量的蛋白样品,是 WB 实验成功必不可少
细胞沉淀后,加入预冷的 RIPA 液(含PMSF),RIPA 液的用量:按照 0.5 mL 每 5×106 细胞计算。轻轻混匀细胞悬液,用振荡器在 4℃ 振荡 15min 以裂解细胞。 用经蒸馏水预冷的橡皮或塑料细胞刮棒将贴壁细胞转移至一离心管中。 于 4℃,10000 rpm 离心裂解液 15 min,迅速转移上清至另一离心管中,弃掉沉淀。 将 Agarose protein A+G 珠子混匀:用预冷的 PBS 洗涤 Agarose protein A+G 珠子两次,并将其用 PBS 调
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