s*******2
发帖数: 598 | 1 最近我在做Western blot试验时，发现我的细胞在用药物CDDP处理48hours 后，Actin
band消失，但是control cells仍然有，原因何在？
Is it means actin is not a good loading control , I need to switch to GAPDH,
Tubulin as a loading control?
Thanks for your suggestions. |
l****b
发帖数: 400 | 2 GAPDH is a glycolysis enzyme and is regulated by many things. In another
words, it is an awful control. Tubulin is better. HSP90 is also good. both
have good antibodies.
But disappearing actin is interesting. worth looking after, don't you think?
Actin
GAPDH,
【在 s*******2 的大作中提到】
: 最近我在做Western blot试验时，发现我的细胞在用药物CDDP处理48hours 后，Actin
: band消失，但是control cells仍然有，原因何在？
: Is it means actin is not a good loading control , I need to switch to GAPDH,
: Tubulin as a loading control?
: Thanks for your suggestions.
|
l***g
发帖数: 19 | |
h******u
发帖数: 131 | 4 解聚也应该有band.
诱导降解或抑制合成。
【在 l***g 的大作中提到】
: 可能是这个药物可以诱导actin的解聚吧
|
s*******2
发帖数: 598 | 5 Thanks for all information. |
s******y
发帖数: 28562 | 6 Actin 是一个非常好的loading control, 也是一个细胞生存需要的关键蛋白之一，能
够使得这个蛋白彻底看不见了是一件非常奇怪的事情。除非细胞被药物处理之后
彻底坏死导致所有actin 都被降解了。你说的那个CDDP是什么药物？
Actin
GAPDH,
【在 s*******2 的大作中提到】
: 最近我在做Western blot试验时，发现我的细胞在用药物CDDP处理48hours 后，Actin
: band消失，但是control cells仍然有，原因何在？
: Is it means actin is not a good loading control , I need to switch to GAPDH,
: Tubulin as a loading control?
: Thanks for your suggestions.
|
A******y
发帖数: 2041 | 7 Are they the same loading? If your lysates don't have any protein left, of
course you won't have any actin band. Btw, don't assume every lab do BCA or
Bradford before western...
Actin
GAPDH,
【在 s*******2 的大作中提到】
: 最近我在做Western blot试验时，发现我的细胞在用药物CDDP处理48hours 后，Actin
: band消失，但是control cells仍然有，原因何在？
: Is it means actin is not a good loading control , I need to switch to GAPDH,
: Tubulin as a loading control?
: Thanks for your suggestions.
|
j******1
发帖数: 1315 | 8 哪个公司的Actin Antibody? Catalog Number? |
w******a
发帖数: 65 | 9 是全细胞裂解液（whole cell lysates）还是Triton extracts 或者RIPA extracts？
我的个人经验，如果是Triton sample （通常用于co-IP），有一种可能性是抽提不完
全，药物刺激F-actin增加导致这一部分的骨架蛋白不溶于Triton lysis buffer。但如
果是WCL或者RIPA sample就要考虑其他的原因。 |
m***m
发帖数: 58 | 10 Re!
48hrs treatment, it might have affected the transcription/translation of
actin or protein degradation in general. Actin is not always stable. I
used to study transcription factors and actin fluctuates a lot. For your
purpose of loading control, I would try all three of them. Tubulin might be
the most stable one.
【在 h******u 的大作中提到】
: 解聚也应该有band.
: 诱导降解或抑制合成。
|
|
|
l**********1
发帖数: 5204 | 11 Co-ask LZ
Is it an inhibitor of mammalian estrogen receptor?
or enhancer of USP-19
If either answer is Bingo,
pls refer
Ogawa M et al. (2011).
17β-estradiol represses myogenic differentiation by increasing ubiquitin-
specific peptidase 19 through estrogen receptor α.
JBC 286(48):41455-65.
HTTP: //www.ncbi.nlm.nih.gov/pubmed/21971047
or
Sundaram P et al. (2009).
USP19-deubiquitinating enzyme regulates levels of major myofibrillar
proteins in L6 muscle cells.
Am J Physiol Endocrinol Metab. 297: E1283-90.
Abstract:
>These studies demonstrate that USP19 modulates transcription of major
myofibrillar proteins and indicate >that the ubiquitin system not only
mediates the increased protein breakdown but is also involved in the >
decreased protein synthesis in atrophying skeletal muscle.
HTTP: //www.ncbi.nlm.nih.gov/pubmed/19773579
【在 s******y 的大作中提到】
: Actin 是一个非常好的loading control, 也是一个细胞生存需要的关键蛋白之一，能
: 够使得这个蛋白彻底看不见了是一件非常奇怪的事情。除非细胞被药物处理之后
: 彻底坏死导致所有actin 都被降解了。你说的那个CDDP是什么药物？
:
: Actin
: GAPDH,
|
s*******2
发帖数: 598 | 12 Cisplatin, 12uM concentration for 48 hour, and I treated in H460 cells.
【在 s******y 的大作中提到】
: Actin 是一个非常好的loading control, 也是一个细胞生存需要的关键蛋白之一，能
: 够使得这个蛋白彻底看不见了是一件非常奇怪的事情。除非细胞被药物处理之后
: 彻底坏死导致所有actin 都被降解了。你说的那个CDDP是什么药物？
:
: Actin
: GAPDH,
|
s*******2
发帖数: 598 | 13 1st antibody from SigmaA 5316, 2nd antibody anti mouse IgG from Cell
Signaling #
7076S, all produced in 2012.
Are these 2 antibodies not match well?
【在 j******1 的大作中提到】
: 哪个公司的Actin Antibody? Catalog Number?
|
s*******2
发帖数: 598 | 14 Same loading, and CDDP treated cells found clear cleaved Caspapse3 band.But
no actin band.
of
or
【在 A******y 的大作中提到】
: Are they the same loading? If your lysates don't have any protein left, of
: course you won't have any actin band. Btw, don't assume every lab do BCA or
: Bradford before western...
:
: Actin
: GAPDH,
|
T****r
发帖数: 4006 | 15 Cisplatin貌似是很久前用的一种抗癌药。
很多常见的house keeping genes,比如actin, tubulin,Gapdh并不是一成不变的，这些
基因也会被调控，包括transcriptional and posttranscriptional的调控，有些细胞
（比如脂肪细胞）只有很少量的actin and tubulin，还是可以继续活着的。 |
y*****i
发帖数: 23 | 16 据我所知，cisplatin是一个化学交联剂。
Mass spectrometry evidence for cisplatin as a protein cross-linking reagent.
http://www.ncbi.nlm.nih.gov/pubmed/21591778
因此猜测是由于cisplatin将actin交联引起条带弥散以至消失。 |
l***g
发帖数: 19 | 17 很奇怪啊，H460好像是肺癌细胞吧。我原来用过不同浓度（0-30uM）的cisplatin处理
过A549细胞36小时，细胞凋亡可以检测到cleaved PARP，beta-actin的条带不会受到影
响的，我可以检测出来。我是用的SDS sample buffer直接裂解的细胞。 |
s*****g
发帖数: 7857 | 18 http://en.wikipedia.org/wiki/Cisplatin
Cisplatin, cisplatinum, or cis-diamminedichloroplatinum(II)[1] (CDDP) (trade
name Cisplatin,brand name Platin marketed by Cadila Healthcare according to
FDA Orange Book) is a chemotherapy drug. It was the first member of a class
of platinum-containing anti-cancer drugs, which now also includes
carboplatin and oxaliplatin. These platinum complexes react in vivo, binding
to and causing crosslinking of DNA, which ultimately triggers apoptosis (
programmed cell death).
Cisplatin是强烈的抗癌药.如果浓度高,很快细胞就翘了.何况48小时处理...也许都
apoptosis光了.
Actin
GAPDH,
【在 s*******2 的大作中提到】
: 最近我在做Western blot试验时，发现我的细胞在用药物CDDP处理48hours 后，Actin
: band消失，但是control cells仍然有，原因何在？
: Is it means actin is not a good loading control , I need to switch to GAPDH,
: Tubulin as a loading control?
: Thanks for your suggestions.
|
A******y
发帖数: 2041 | 19 How do you determine your loading?
But
【在 s*******2 的大作中提到】
: Same loading, and CDDP treated cells found clear cleaved Caspapse3 band.But
: no actin band.
:
: of
: or
|
s******y
发帖数: 28562 | 20 我对你们用的药不熟悉，刚才上网查了一下，这个药物对F-actin 有已知的破坏作用，http://www.nature.com/cdd/journal/v5/n7/abs/4400392a.html
但是对monomeric actin 到还没有报道过有什么效果。
Actin 本身是一个非常稳定的蛋白，half life 超过24小时。如果居然彻底消失了是个
很蹊跷的事情。
对于你们的实验，首先，先试用其他loading control, 不要耽搁你们的实验了。
然后对于actin 消失这件事情，再采用其他方法来测试看看是不是你们的细胞系
有非常强的杀灭作用，或者象其他同学建议的那样，是不是转换成其他类型的细胞了。
另外就是注意一定要有positive and negative control during western blot.
前一阵子我实验室里的人也嚷嚷说actin band 看不见了，后来我去仔细查了一下，其
实就是二抗坏掉了而已，换一个新的就什么都出来了。
【在 s*******2 的大作中提到】
: Cisplatin, 12uM concentration for 48 hour, and I treated in H460 cells.
|
|
|
c******d
发帖数: 647 | 21 second this,cisplatin就是起交联作用，重要的作用对象就是DNA
这种药物作用很强，12uM,48小时，到底细胞死了多少呀，
我觉得恐怕没多少剩下了，用过2uM处理48小时，细胞没剩多少，但actin作为loading
control没啥变化
trade
to
class
binding
【在 s*****g 的大作中提到】
: http://en.wikipedia.org/wiki/Cisplatin
: Cisplatin, cisplatinum, or cis-diamminedichloroplatinum(II)[1] (CDDP) (trade
: name Cisplatin,brand name Platin marketed by Cadila Healthcare according to
: FDA Orange Book) is a chemotherapy drug. It was the first member of a class
: of platinum-containing anti-cancer drugs, which now also includes
: carboplatin and oxaliplatin. These platinum complexes react in vivo, binding
: to and causing crosslinking of DNA, which ultimately triggers apoptosis (
: programmed cell death).
: Cisplatin是强烈的抗癌药.如果浓度高,很快细胞就翘了.何况48小时处理...也许都
: apoptosis光了.
|
s*******2
发帖数: 598 | 22 See below 1997 paper
Biochem J. 1997 Apr 1;323 ( Pt 1):233-7.
Actin is cleaved during constitutive apoptosis.
Brown SB, Bailey K, Savill J.
SourceDivision of Renal and Inflammatory Disease, Department of Medicine,
University Hospital, Nottingham NG7 2UH, UK.
Abstract
Proteases play an important role in the programme of cell death by apoptosis
but little is known of the substrates cleaved, particularly in constitutive
models of this type of cell death. Neutrophils spontaneously undergo
apoptosis in culture without requiring external stimuli. During this process
we found biochemical and immunochemical evidence for the cleavage of
membrane-associated actin, a component of the cytoskeleton that links
polymerized actin to the plasma membrane. Cleavage occurred at a single site
at the N-terminus, between residues Val43-Met44, a site devoid of a
consensus motif for cleavage by cysteine proteases of the interleukin-1beta-
converting enzyme (ICE)-family. Whereas actin cleavage and nuclear/cell
surface markers of apoptosis were co-ordinately diminished by zVAD-fmk, an
inhibitor of the ICE-like family of proteases, only acetyl-leucyl-
leucylnormethional, an inhibitor of calpains, was capable of completely
inhibiting actin cleavage. Our results suggest that actin is not a direct
substrate for the ICE-like family of proteases. By disabling the
cytoskeleton, actin cleavage may be an important component in the capacity
of apoptosis to reduce the injurious potential of neutrophils.
PMID:9173887[PubMed - indexed for MEDLINE] PMCID:PMC1218300 |
s*******2
发帖数: 598 | 23 We just got new 2nd anti-mouse IgG from cell signaling 2-3 months ago, and
even I used 1/1000 as 1st antobody, 1/2000 as 2nd antibody on 1/16/13
experiement, too strong for actin, but no band for drug tretament group.
，http://www.nature.com/cdd/journal/v5/n7/abs/4400392a.html
【在 s******y 的大作中提到】
: 我对你们用的药不熟悉，刚才上网查了一下，这个药物对F-actin 有已知的破坏作用，http://www.nature.com/cdd/journal/v5/n7/abs/4400392a.html
: 但是对monomeric actin 到还没有报道过有什么效果。
: Actin 本身是一个非常稳定的蛋白，half life 超过24小时。如果居然彻底消失了是个
: 很蹊跷的事情。
: 对于你们的实验，首先，先试用其他loading control, 不要耽搁你们的实验了。
: 然后对于actin 消失这件事情，再采用其他方法来测试看看是不是你们的细胞系
: 有非常强的杀灭作用，或者象其他同学建议的那样，是不是转换成其他类型的细胞了。
: 另外就是注意一定要有positive and negative control during western blot.
: 前一阵子我实验室里的人也嚷嚷说actin band 看不见了，后来我去仔细查了一下，其
: 实就是二抗坏掉了而已，换一个新的就什么都出来了。
|
g**c
发帖数: 144 | 24 换一抗试试看？随便找谁借点儿别的公司的。也许药把这个公司的 antibody 所识别的
actin 的 epitope 给搞得认不出了。。。或者甚至直接用药处理 control cell lysa
te，看看 actin 条带会不会消失。
Actin
GAPDH,
【在 s*******2 的大作中提到】
: 最近我在做Western blot试验时，发现我的细胞在用药物CDDP处理48hours 后，Actin
: band消失，但是control cells仍然有，原因何在？
: Is it means actin is not a good loading control , I need to switch to GAPDH,
: Tubulin as a loading control?
: Thanks for your suggestions.
|
s*******2
发帖数: 598 | 25 Updated: I tried using Santa Cruz GAPDH(anti-rabbit) as loading control on 1
/24 and 1/25, it is fine. |
