T*****n
发帖数: 897 | 1 我们做microarray发现了一些基因表达降低，随后用real time PCR 也得到确认。但我
们试图用western blot及ELISA的方法去证明蛋白表达也降低时却遇到麻烦，因为
western和ELISA显示蛋白表达正常。有点想不通。大家遇到过这样的情况吗？ |
a*****x
发帖数: 901 | 2 Could be slow turnover proteins |
T*****n
发帖数: 897 | 3 大家遇到过这种情况吗？我听人说:usually, the results of WB match with those
of real time PCR. 但在这里好像不是这么回事。 |
l******g
发帖数: 1145 | 4 很正常，我碰到过某蛋白mRNA调控上升明显，westernblot出来是急剧减少，还以为实
验哪出问题了。troubleshooting了很久，发现就是这么回事。。。 |
D******9
发帖数: 2665 | 5 do you know the half life-time of your interested protein? Also there could
be other post-transcriptional mechanisms involved. |
T*****n
发帖数: 897 | 6 Thank you for your reply. Yes, you are right,but how often the situations
you described will happen. I heard that usually the WB results match with
real time PCR. Is that right?
could
【在 D******9 的大作中提到】
: do you know the half life-time of your interested protein? Also there could
: be other post-transcriptional mechanisms involved.
|
i***l
发帖数: 1656 | |
l**********1
发帖数: 5204 | 8 Please refer
Harmonics of Circadian Gene Transcription in Mammals
PLoS Genet. 2009 April; 5(4): e1000442.
Published online 2009 April 3
Validation experiments show that 12-hour oscillatory transcripts occur in several other peripheral tissues
as well including heart, kidney, and lungs. These harmonics are lost ex vivo, as well as under restricted
feeding conditions. Taken in sum, these studies illustrate the importance of time sampling with respect to
multiple testing, suggest caution in use of autonomous cellular models to study clock output
//www.ncbi.nlm.nih.gov/pmc/articles/PMC2654964/
and
http://www.mitbbs.com/article_t1/Biology/31646749_0_7.html
[回复]
0
[ 121 ]
发信人: abq (NM), 信区: Biology
标 题: Re: 哪位能科普一下ZHUANG的单分子荧光
发信站: BBS 未名空间站 (Thu Mar 22 04:53:05 2012, 美东)
Xiao-Liang Sunny Xie
his work:
在Harvard开始的方向是在单细胞+单分子水平上观察细菌基因的表达和调控，主要是分
析Transcription和Translation的随机性。有一个结果特别有意思，就是在大肠杆菌里
面，任何一个时刻mRNA的copy number和对应蛋白质的copy number完全没有任何相关性
。这个方向成就了好几个现在转做系统生物学的人。
more literatures:
//www.ncbi.nlm.nih.gov/pubmed/18369419
//www.ncbi.nlm.nih.gov/pubmed/20934868
//www.ncbi.nlm.nih.gov/pubmed/22419973
//www.ncbi.nlm.nih.gov/pubmed/18510756
//journals.cambridge.org/action/displayAbstract?fromPage=online&aid=8025948
HTTPS//www.rocq.inria.fr/bang/JC/JC4MMNP2009.pdf
HTTPS//www.rocq.inria.fr/bang/JC/JCarticles.html
【在 T*****n 的大作中提到】
: 大家遇到过这种情况吗？我听人说:usually, the results of WB match with those
: of real time PCR. 但在这里好像不是这么回事。
|
l**********1
发帖数: 5204 | 9 Just try
ChIP-RNA
Protocol:
//webdoc.nyumc.org/nyumc/files/sun-lab/attachments/CPMB.Ch.27.RNA.Protein.pdf
alternatively:
try below steps:
IN VIVO sampling from cell lines/KO^+ + or KO^-- Mouse
separated to half and half
with half do mRNA microarray and real time PCR
then await 12 hr another half do WB. NB: this 12 hr should be cultured at
same cell line/Mouse
more:
//sun-lab.med.nyu.edu/our-group/lab-login/experimental-protocols
PS: if your job worked later, please transfer BAOZI one to my mitbbs financial account,
W-E-I-B-I ten .
【在 T*****n 的大作中提到】
: 我们做microarray发现了一些基因表达降低，随后用real time PCR 也得到确认。但我
: 们试图用western blot及ELISA的方法去证明蛋白表达也降低时却遇到麻烦，因为
: western和ELISA显示蛋白表达正常。有点想不通。大家遇到过这样的情况吗？
|
t*****2
发帖数: 213 | 10 usually the protein does not match mRNA . It's normal.
【在 T*****n 的大作中提到】
: 大家遇到过这种情况吗？我听人说:usually, the results of WB match with those
: of real time PCR. 但在这里好像不是这么回事。
|
a***y
发帖数: 19743 | 11 there is always exceptions!
【在 T*****n 的大作中提到】
: Thank you for your reply. Yes, you are right,but how often the situations
: you described will happen. I heard that usually the WB results match with
: real time PCR. Is that right?
:
: could
|
