试剂名称 | 体积 | 保存条件 |
Lysis buffer | 30ml | 4 ℃ |
10*Wash buffer | 50ml | 4 ℃ |
1*SDS 样品缓冲液 | 1ml | 4 ℃ |
Protein A 琼脂糖珠 | 250ul | 4 ℃ |
Protein G 琼脂糖珠 | 250ul | 4 ℃ |
A. 制备细胞裂解物
1. 吸干培养基。添加含有调节分子的新鲜培养基,使其在预定的时间内对细胞进行处理。
2. 收集细胞,去除培养基后用冰预冷的 1 X PBS 洗涤细胞一次。
3. 去除 PBS 并在每个细胞板上(10 cm)加入 0.5 ml 冰预冷的 Lysis buffer,并在冰上孵育至少5分钟。
4. 从板上刮下细胞,把提取物转移至微量离心管。置于冰上。
5. 在冰上进行 3 次超声破碎,每次 5 秒。(PS:超声条件需根据液体量、探头直径以及最大功率调整。以200W功率超声仪器为例,用 3 mm 的探头。我们一般推荐使用 35%-40% 的最大功率进行 3 次超声脉冲, 每次超声之间停顿 10 秒。)
6. 在 4 °C,在 14,000 x g 条件下,微量离心10分钟,将上清转移到新管中。上清液即为细胞裂解物。如有必要,裂解物可保存在 -80 °C。
注:细胞裂解物制备好之后,可先进行蛋白免疫印迹法检测,以此来确定细胞裂解物里存在目标蛋白。
B. 免疫沉淀法
a) 细胞裂解物预清除(可选步骤)
1. 向 500 μl(含总蛋白 200 -1000 ug)细胞裂解物中添加 5 μl Protein A 和 5 μl Protein G。
2. 在 4 °C 下,旋转孵育 30 - 60 分钟。
3. 4°C 条件下 12000g 离心1分钟,保留上清,待用。
b) 抗原抗体结合
1. 在新的离心管内加入 500 μl (含总蛋白 200 – 1000 ug)细胞裂解物(可以是预清洗后的细胞裂解物)。
2. 加入目标蛋白的单克隆/多克隆抗体(1 – 5 μg)。
3. 同时采用非特异免疫的同源抗体作为对照。
4. 在 4 °C 轻柔混合过夜。
c) 免疫复合物沉淀
1. 抗体孵育过夜后,加入 5 μl Protein A 和 5 μl Protein G。
2. 在 4 °C 温和地混匀1- 3小时或过夜。
3. 12,000 g 离心1 分钟,保留沉淀。
4. 使用 0.5 ml 1*Wash buffer 清洗沉淀,12,000 g 离心1分钟,保留沉淀。
5. 重复第4步,共计清洗3次。
注:清洗,去上清的时候需要注意避免吸走填料
C. 用蛋白免疫印迹法进行分析
1. 在 20 - 40 μl 1*SDS 样品缓冲液中重悬沉淀物,涡旋振荡,然后再微量离心30秒,以使附着管壁的珠子和液体到管底。
2. 将样品加热至 95 - 100 °C 并持续2 - 5分钟,然后在 14,000 g下瞬时离心1分钟,取上清液。
3. 将样品(15 – 30 μl)上样到 SDS-PAGE 凝胶上。
4. 用蛋白质印迹法分析样品。
注意:Protein A和Protein G的基质为20%乙醇,较易挥发,使用时应注意密封,如出现挥发情况,可使用20%乙醇重悬。
- 实验方法 实验条件
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- 如果我后续是想要跑native胶,不做变性电泳,不能用试剂盒里的1*SDS样本缓冲液,我应该用什么替代呢?
- 用不含有SDS的WB上样缓冲液,但不经过变性,蛋白能否解离下来,我们无法确定,不能保证您后续的实验效果
- 如果我的细胞裂解物里面蛋白含量高于试剂盒要求的总蛋白含量,我应该用什么试剂进行稀释呢?
- 常规用PBS稀释即可
- lysis buffer和PMSF怎么使用?
- 根据使用量,取每1ml lysis buffer加入10ul PMSF(100mM),使PMSF的最终浓度为1mM,混匀备用 (PMSF现用现加)。
- protein A和protein G beads怎么选?
- 各取5ul。
- 超声破碎冰上操作是必须的吗?4℃条件能否微调?
- 建议冰上操作,4℃可以微调。
- beads出现吸不出来的情况,怎么处理?
- 吸之前需注意枪头要剪一下再吸,如若beads吸不上来,可加入等体积的20%乙醇混匀后再吸。
- 如果没有超声破碎仪的话,能不能用酶解的办法破碎细胞?
- 酶解和超声都可以,酶按照实验室正常使用浓度配制即可。
- 实验的时候能抗体beads一起加吗?还是说先加抗体再加beads?
- 都可以,但我们建议参考产品说明书使用,先加抗体再加beads。
- 10*Wash buffer如何稀释?一定要使用配套的吗?
- dd水稀释即可,最终浓度是1 * washing buffer 即可。
- 怎么看出现的条带结果?
- 比较关注的是三条泳道,input和IgG以及检测互相作用的泳道,理论上input有一条目的条带, IgG没有条带。如果input没有条带,说明在进行免疫共沉淀之前没有进行WB验证,建议先验证之后 再重新操作。若IgG阴性对照出现条带,是不正常的,应是受重链和轻链的影响,说明使用的二抗 不正确, 建议更换特异性更强的二抗.
- 免疫共沉淀试剂盒裂解液能用于组织吗?比例又是如何?
- 可用于组织,要先将组织研磨成匀浆,比例和细胞比例一致。
- 阴性对照lgG组后续WB有条带一般是什么原因?
- 首先需要排除lgG和后续WB一抗同源导致的轻重链的干扰条带(25kd和50kd附近);再者建议参考本试剂盒免疫沉淀法“可选步骤”以排除Protein A/G的非特异性结合;再就是考虑lgG的问题可以换用其他品牌lgG尝试。
- 步骤上的可选步骤能否省略?
- 可选步骤为预处理,目的为去除样本中可与protein A/G非特异结合的蛋白质,减少干扰,如CoIP实验中发现有较多杂带,可考虑选做;但应注意,选择此步骤后,试剂盒中protein A和protein G对应实验样本数减半。
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