应《网络安全法》要求，自2017年10月1日起，未进行实名认证将不得使用互联网跟帖服务。为保障您的帐号能够正常使用，请尽快对帐号进行手机号验证，感谢您的理解与支持！

24小时热门版块排行榜    

小木虫论坛-学术科研互动平台 » 生物医药区 » 生物科学 » 蛋白电泳/WB » western blot小分子检测不出
71/1返回列表
查看: 2977  |  回复: 6
只看楼主 @他人 存档 新回复提醒 (忽略) 收藏    在APP中查看

lianchen1218

金虫 (正式写手)

[求助] western blot小分子检测不出 已有3人参与

最近在做细胞色素c cytochrome c的western blot,可是总是检测不出来，现在是和一个月之前同样的细胞系，同样的处理，同样的操作步骤，就是所用的东西都一样，可是就是检测不出来，之前都可以检测出来。
我的电泳条件是
15%的胶
60-70V浓缩胶，分离胶就100-120V，蛋白没有跑出来
转膜，4度
120V，90min; 110v,60min; 110V, 100min都试过
到丽春红染色的时候15-10KD的条带实验组就几乎没有，可是同样的一张膜上，阳性对照组在15-10KD又有明显的条带，而且上样量都一样。
缓冲液都没超过三次使用。
封闭5%牛奶，1h
二抗也是1h
显影的时候，有时候阳性对照都没有细胞色素C，但是有时候又有，不过实验组就一直没有。
我的细胞处理是
收集后离心，提取沉淀，用细胞分离液含0.01%digitonin，常温，15min，目的是分离细胞溶胶和其余的细胞器，因为cytochrome c是从线粒体释放到细胞溶胶的。
完全一样的操作，可是现在就是怎么都检测不到细胞色素c，一个月了，耽误了不少时间。请大家指导一下，可能是哪里出了问题？
拜谢了！
回复此楼

» 猜你喜欢

» 本主题相关商家推荐: (我也要在这里推广)

» 本主题相关价值贴推荐，对您同样有帮助:
勇敢一点，去充实自己
1楼 2014-12-09 02:33:03
已阅   回复此楼   关注TA 给TA发消息 送TA红花 TA的回帖

fuyuandj86

版主 (著名写手)

己所不欲，勿施于人

【答案】应助回帖

感谢参与，应助指数 +1
重新配一管一抗过夜孵育
赞一下 回复此楼
The doer of good becomes good, the doer of evil becomes evil.
2楼2014-12-09 03:02:25
已阅   回复此楼   关注TA 给TA发消息 送TA红花 TA的回帖

lianchen1218

金虫 (正式写手)
引用回帖:
2楼: Originally posted by fuyuandj86 at 2014-12-09 03:02:25
重新配一管一抗过夜孵育

尝试过了，还是不行，我现在就怀疑自己的样品处理有问题。
赞一下 回复此楼
勇敢一点，去充实自己
3楼2014-12-11 23:59:17
已阅   回复此楼   关注TA 给TA发消息 送TA红花 TA的回帖

嘿小杨同志

金虫 (小有名气)

【答案】应助回帖

楼主 你是半干法转膜吧？ 如果是的话，多转一会，蛋白大小不同转膜的时间不同，小蛋白快点，大蛋白慢。
赞一下 回复此楼
4楼2014-12-19 11:22:45
已阅   回复此楼   关注TA 给TA发消息 送TA红花 TA的回帖

needle_ld

至尊木虫 (正式写手)

木虫学者

【答案】应助回帖

How do you make transfer buffer?
do you have methanol? how much?
I would recommend 20%mathanol in the transfer buffer. or, maybe you can decrease your time for transfer?

Where do you get the first antibody? how do you keep the stock? if you have the bands some time ago but not now, maybe your antibody is too old? especially if the antibody is kept in fridge but not freezer.
you only do WB for cytochrome C? How about other targets? do you have any problem in house keeping (like GAPDH, tubulin)? if so, maybe the problem is more general.
赞一下 回复此楼
平等观诸法，悲心救世间。解彼真实性，得佛智慧光。
5楼2014-12-19 13:28:36
已阅   回复此楼   关注TA 给TA发消息 送TA红花 TA的回帖

lianchen1218

金虫 (正式写手)
引用回帖:
5楼: Originally posted by needle_ld at 2014-12-19 13:28:36
How do you make transfer buffer?
do you have methanol? how much?
I would recommend 20%mathanol in the transfer buffer. or, maybe you can decrease your time for transfer?

Where do you get the fir ...

Thank you for your suggestion.
First, I add 20% methanol in the transfer buffer. And I already decreased the time to 1 h, 110V. And used the new prepared primary antibody, increased loading to 40ug, still nothing.
Second, antibody are kept in fridge. Because the others can detect the cytochrome c with the same primary antibody, so I don't think it's the problem.
I also do other targets, GAPDH works well. Except cytochrome c, all the other staff work well.
赞一下 回复此楼
勇敢一点，去充实自己
6楼2014-12-20 06:35:55
已阅   回复此楼   关注TA 给TA发消息 送TA红花 TA的回帖

needle_ld

至尊木虫 (正式写手)

木虫学者
引用回帖:
6楼: Originally posted by lianchen1218 at 2014-12-19 17:35:55
Thank you for your suggestion.
First, I add 20% methanol in the transfer buffer. And I already decreased the time to 1 h, 110V. And used the new prepared primary antibody, increased loading to 40ug ...

OK. Then you are correct. The problem is most likely your sample prep.
for cells, it is easy to get lysate. maybe you can try different RIPA. I am using RIPA with NP-40 and it works well.
also, it would be better if you can isolate the mitochondria and then make the lysate. it is not difficult to do so provided that you may want to have dounce which is a little bit less tight than normal homogenizer. then you can separate the lysate using percoll ultracentrifuge. you can find the protocol for MAM preparation in Nature protocol (2009). you don't need to get the MAM and the mitochondria is very easy by product of the protocol.
the other point regarding WB, don't rely on the molecular weight marker. try to terminate the SDS-PAGE earlier. 15% gel should be ok. I can do with it by 10% gel sometimes. we are using cell signaling antibody which works well.
赞一下 回复此楼
平等观诸法，悲心救世间。解彼真实性，得佛智慧光。
7楼2014-12-20 13:22:13
已阅   回复此楼   关注TA 给TA发消息 送TA红花 TA的回帖
相关版块跳转 我要订阅楼主 lianchen1218 的主题更新
71/1返回列表
普通表情 龙 兔 虎 猫
信息提示
关闭
请填处理意见
关闭