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细胞核蛋白和胞浆蛋白制备试剂盒 Nuclear-Cytosol Extraction Kit P1200厂家直销，提供OEM定制服务！
- 产品详情
- 说明书下载
- 引用文献
描述：用于从哺乳动物组织和培养细胞中提取核蛋白和/或胞浆蛋白，提取制备过程简便。制备的核蛋白和胞浆蛋白能保持天然活性，并且纯度高。提取的蛋白适于转录因子活性分析、凝胶阻滞实验(gel shift assay, EMSA)、免疫共沉淀、酶活性测定，也适于Western Blot实验。
组份：
|
试剂盒组成 |
50次制备 |
100次制备 提取 |
储存条件 |
|
Cytosol Extraction Buffer A (CEB-A) |
25 ml |
50 ml |
4 oC, 1 year |
|
Cytosol Extraction Buffer B (CEB-B) |
1.5 ml |
2×1.5 ml |
4 oC, 1 year |
|
Nuclear Extraction Buffer (NEB) |
5 ml |
10 ml |
4 oC, 1 year |
温馨提示：
1. 操作应在冰浴中进行，试剂需提前预冷。
2. 根据大致的细胞数，或大致估计离心后的细胞体积(Packed cell volume,PCV)来确定试剂的加入量。PCV因细胞类型不同而有所差异。细胞数、PCV
与试剂加入量之间的对应关系，参见下表：
|
培养皿直径 |
35 mm or或1孔六孔板 |
60 mm |
10 cm |
|
培养皿面积 |
10 cm2 |
30 cm2 |
75 cm2 |
|
细胞计数 |
5 ×105 |
1 ×106 |
5 ×106 |
|
相当于PCV(ul) |
5 |
10~20 |
50~100 |
|
加入CEB-A (ul) |
25 |
50~100 |
300~500 |
|
加入CEB-B (ul) |
1.2~1.5 |
3~6 |
3~6 |
|
加入NEB (ul) |
5~10 |
20 |
100 |
操作步骤:
1.裂解：(1) 培养细胞，PBS洗涤细胞两次，根据细胞计数，或者估计离心后的细胞体积PCV。每1 ×106个细胞加入50~100ul的CEB-A，刮下细胞转移
至预冷的1.5ml离心管；或者每体积PCV细胞加入5倍PCV体积的CEB-A (即10~20 ul PCV的细胞加50~100ul的CEB-A)，剧烈振荡重悬。
裂解: (2) 组织样本，精确称重后剪成小块，PBS洗涤。通常每10mg动物组织相当于1×106个细胞，需加入50~100ul CEB-A，参照上表按比例加入。
在冰上使用玻璃匀浆器匀浆组织，通常需要20~40次上下抽动匀浆，弃去筋膜组织。切记勿用高速电动匀浆器或超声破碎组织避免破坏细胞器结构。
2.裂解产物转移至预冷的1.5ml离心管，剧烈振荡30秒，冰浴10~15min，期间每5分钟振荡15秒。
3.可选步骤：根据步骤2裂解物体积，加入1/20体积的CEB-B，振荡10秒，冰浴1min (加入CEB-B可去除部分核膜蛋白，适于制备高纯度的核蛋白用
于EMSA凝胶阻滞实验等，但可使胞浆蛋白出现很少量的膜蛋白污染。若制备高纯度胞浆蛋白可跳过此步骤。若制备核蛋白仅用于普通的Western
Blot目的，则无须高纯度核蛋白，也可省略此步骤。
4. 胞浆蛋白组分的制备：将步骤2或步骤3的上清，12000g 4oC 离心5min，勿触动沉淀，将上清转移到新管，此即胞浆蛋白组分，可立即使用或
−20~−70ºC保存。
5. 胞核粗提组分的制备：取第步骤4的原离心管，12000g 4oC瞬时离心，尽量吸除上清，保留沉淀。加入100ulCEB-A和5ulCEB-B，振荡10秒，
重悬沉淀，冰浴1min，
1000g 5min，弃上清。再次加入100ulCEB-A和5ulCEB-B重悬沉淀冰浴1min,1000g 5min，尽弃上清，保留沉淀。
6. 胞核蛋白的制备：加50~100ul预冷NEB重悬步骤5的离心沉淀，剧烈振荡15秒，冰上30min，期间每10min振荡15秒。12000g 5min，上清液含核
蛋白成分，其中含有25%的甘油；可立即使用或−20~−70ºC保存。
说明：
1. 严格按照说明书操作，每106个细胞大约得到50-75ug蛋白。2. 裂解时间是重要的，过短则细胞裂解不全导致蛋白产率低，裂解时间长则导致胞浆
蛋白有核蛋白污染。3.只需对提取的胞浆蛋白定量(Braford或BCA法)，核蛋白纯度高但含量低，无需定量，对于同一个制备，核蛋白与胞浆蛋白的量
于相平行。4. 上述制备的蛋白样品与2xSDS-PAGE混合即可上样电泳。5. 切记采用手动玻璃匀浆器，勿用高速电动匀浆器或超声破碎组织细胞，避免
破坏细胞器结构，导致分离的组分污染。玻璃匀浆器须配套选用间隙严紧的研杵，其特征是将研杵插入匀浆器套管后，提起研杵而套管不会脱落。
正确匀浆是先下压旋转研杵挤破组织，然后上下缓慢推拉研杵破碎细胞。组织样品制备效果不如细胞样品。
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