C****c
发帖数: 1964 | 1 现在用的是actin，经常很脏，而且loading variation很大，大家都用什么loading
control呢？有用tubulin的吗？求推荐一个干净的tubulin antibody？谢谢！ |
z*********8
发帖数: 1203 | |
C****c
发帖数: 1964 | |
l****b
发帖数: 400 | 4 是你的sample脏，不是actin脏。你的lysate如果是干干净净的，没有脂类混在里头，
用tubulin和actin的效果是差不多的。反之，用啥都有smear。 |
y****i
发帖数: 2194 | 5 abcam的单抗tubulin非常好用
【在 C****c 的大作中提到】
: 现在用的是actin，经常很脏，而且loading variation很大，大家都用什么loading
: control呢？有用tubulin的吗？求推荐一个干净的tubulin antibody？谢谢！
|
A******y
发帖数: 2041 | 6 How do you normalize your loading? If you use protein concentrations, you
need to dilute some lysate in 2% SDS and do a BCA assay. It is likely your
loading variant is big. |
C****c
发帖数: 1964 | 7 这个我也知道，不过肝脏的脂肪很难完全去掉，因为太多了
【在 l****b 的大作中提到】
: 是你的sample脏，不是actin脏。你的lysate如果是干干净净的，没有脂类混在里头，
: 用tubulin和actin的效果是差不多的。反之，用啥都有smear。
|
C****c
发帖数: 1964 | 8 Usually I weigh the tissues. I tried to do BCA before, but it is not very
accurate. Does it work well when you dilute it in 2% SDS and do BCA assay?
Thanks!
your
【在 A******y 的大作中提到】
: How do you normalize your loading? If you use protein concentrations, you
: need to dilute some lysate in 2% SDS and do a BCA assay. It is likely your
: loading variant is big.
|
z*********8
发帖数: 1203 | 9 我也做的tissue，我觉得normalize到重量上很不可靠啊，因为有些tissue lysis的效
率感觉不太一样，我个人觉得normalize protein input 用bradford倒是还不错的感觉。
【在 C****c 的大作中提到】
: Usually I weigh the tissues. I tried to do BCA before, but it is not very
: accurate. Does it work well when you dilute it in 2% SDS and do BCA assay?
: Thanks!
:
: your
|
l****b
发帖数: 400 | 10 It is difficult to remove the fat, but not impossible. Spin first, then use
gentle vacuum to remove the top layer, which contains mostly lipids. Repeat
the process if necessary. The remaining lysates can be further diluted to
reduce the lipid concentration. Since liver samples generally have plenty of
proteins so diluting them would be OK. It is a pain in the neck but with
this method my liver western blots, even with samples from high fat fed mice
, look perfectly fine.
【在 C****c 的大作中提到】
: 这个我也知道，不过肝脏的脂肪很难完全去掉，因为太多了
|
C****c
发帖数: 1964 | 11 用重量需要每一步所有的sample都要很consistent。下次我再试试dilute到SDS在做BCA。
觉。
【在 z*********8 的大作中提到】
: 我也做的tissue，我觉得normalize到重量上很不可靠啊，因为有些tissue lysis的效
: 率感觉不太一样，我个人觉得normalize protein input 用bradford倒是还不错的感觉。
|
C****c
发帖数: 1964 | 12 Thanks a lot for the tips.
use
Repeat
of
mice
【在 l****b 的大作中提到】
: It is difficult to remove the fat, but not impossible. Spin first, then use
: gentle vacuum to remove the top layer, which contains mostly lipids. Repeat
: the process if necessary. The remaining lysates can be further diluted to
: reduce the lipid concentration. Since liver samples generally have plenty of
: proteins so diluting them would be OK. It is a pain in the neck but with
: this method my liver western blots, even with samples from high fat fed mice
: , look perfectly fine.
|
f*******e
发帖数: 354 | 13 脏可能是的样品要多多离心几次了，但是variation我觉得避免不了，参考northern，
组织样品loading control变化非常大
【在 z*********8 的大作中提到】
: 我也觉得actin的wb好脏啊，同求！
|
w*******d
发帖数: 396 | 14 我试过下面的方法。材料是小鼠肝脏。供参考。
加PBS，匀浆（homogenize)，离心，裂掉RBC，加PBS，离心，用PBS洗一次，这时的上
清不再是浑浊的了，用RIPA buffer裂解细胞，离心，收集上清，BCA测蛋白浓度，
normalize. |
