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Cannot get beta-actin, Why?
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Cannot get beta-actin, Why?# Biology - 生物学
c*i
1
弱问 假如我有现金若干和支票若干 应该怎么给SMART CASH存钱啊?
用checking account #去任何银行都可以存? 没手续费?
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l*r
2
换了个router,连内网上的蓝光原盘,看了一会儿就开始卡了。
找到个解决办法,就是打开无线,然后在关掉(原盘都是gigabit网线连的)。就可以
解决,但关机后再开,问题又有了。 你们的有这个问题吗?
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E*1
3
I cannot get beta-actin, other proteins is ok.
I tried several actin antibodies, none works.
I am thinking maybe semi-dry transfer too long, but 11-40KD marker still
there.
Whats happened?
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a*g
4
转账
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z*0
5
无线连接不可靠啊!
我最近买了个最便宜的600F,用powerline有线连接,看内网的高清电影或国内电视和
视频都不卡,就是开始时需要缓冲几秒钟。

【在 l*****r 的大作中提到】
: 换了个router,连内网上的蓝光原盘,看了一会儿就开始卡了。
: 找到个解决办法,就是打开无线,然后在关掉(原盘都是gigabit网线连的)。就可以
: 解决,但关机后再开,问题又有了。 你们的有这个问题吗?

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s*y
6
You mean you cannot detect beta-actin by WB on your membrane?

【在 E*****1 的大作中提到】
: I cannot get beta-actin, other proteins is ok.
: I tried several actin antibodies, none works.
: I am thinking maybe semi-dry transfer too long, but 11-40KD marker still
: there.
: Whats happened?

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W*e
7
支票可以用手机存

【在 c*******i 的大作中提到】
: 弱问 假如我有现金若干和支票若干 应该怎么给SMART CASH存钱啊?
: 用checking account #去任何银行都可以存? 没手续费?

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l*r
8
内网高清,无线看720p没问题,1080p或蓝光盘就不行了。

【在 z****0 的大作中提到】
: 无线连接不可靠啊!
: 我最近买了个最便宜的600F,用powerline有线连接,看内网的高清电影或国内电视和
: 视频都不卡,就是开始时需要缓冲几秒钟。

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E*1
9
yes.
I am confused
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d*t
10
手机或者mail

【在 c*******i 的大作中提到】
: 弱问 假如我有现金若干和支票若干 应该怎么给SMART CASH存钱啊?
: 用checking account #去任何银行都可以存? 没手续费?

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z*0
11
1080p或蓝光盘没试过,估计也不行。但看网上视频足够了。

【在 l*****r 的大作中提到】
: 内网高清,无线看720p没问题,1080p或蓝光盘就不行了。
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s*y
12
什么样品?用什么胶跑的?转到了什么膜上面?
你有没有用Ponceau S 染染看蛋白是否转过去了?
如果你的样品里有actin 的话,即使转膜不完全,不可能一点都看不到的,
如果什么都看不见,那就有几个可能:
1。 你的二抗坏掉了,或者你的WB reagnet 有问题。 这个是最有可能的。
2。你跑的样品根本就不是普通的真核细胞的样品。

【在 E*****1 的大作中提到】
: yes.
: I am confused

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i*l
13
try this:Sigma no. A2066 works well for me

【在 E*****1 的大作中提到】
: I cannot get beta-actin, other proteins is ok.
: I tried several actin antibodies, none works.
: I am thinking maybe semi-dry transfer too long, but 11-40KD marker still
: there.
: Whats happened?

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E*1
14
regular protein, I got it before
But I cannot get it now
WB reagnet? What is that?

【在 s******y 的大作中提到】
: 什么样品?用什么胶跑的?转到了什么膜上面?
: 你有没有用Ponceau S 染染看蛋白是否转过去了?
: 如果你的样品里有actin 的话,即使转膜不完全,不可能一点都看不到的,
: 如果什么都看不见,那就有几个可能:
: 1。 你的二抗坏掉了,或者你的WB reagnet 有问题。 这个是最有可能的。
: 2。你跑的样品根本就不是普通的真核细胞的样品。

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E*1
15
thanks. Dear
I think I use this one too but I cannot get ....55555

【在 i***l 的大作中提到】
: try this:Sigma no. A2066 works well for me
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s*y
16
WB reagent,
比方说你的二抗 (这个是最可能出问题的),
或者chemifluorescent reagents,
buffer (check your PBS or TBS buffer pH)
Sodium Azide (did you mistakenly put sodium azide in your buffer anywhere?)

【在 E*****1 的大作中提到】
: regular protein, I got it before
: But I cannot get it now
: WB reagnet? What is that?

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E*1
17
But why I can get others except actin

【在 s******y 的大作中提到】
: WB reagent,
: 比方说你的二抗 (这个是最可能出问题的),
: 或者chemifluorescent reagents,
: buffer (check your PBS or TBS buffer pH)
: Sodium Azide (did you mistakenly put sodium azide in your buffer anywhere?)

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s*y
18
你是说其他带都能显出来,就是actin 看不见?

【在 E*****1 的大作中提到】
: But why I can get others except actin
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D*a
19
你做你以前做过的WB样品能不能看到带?
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E*1
20
Yes,I can before

【在 D*a 的大作中提到】
: 你做你以前做过的WB样品能不能看到带?
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E*1
21
Yes. It is true

【在 s******y 的大作中提到】
: 你是说其他带都能显出来,就是actin 看不见?
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s*y
22
如果其他带都能看见(你说的那些什么size marker 那个可不算数),
而且用ponceau S染色后有都能看见蛋白的带的话,看不见actin 那真是见鬼了

【在 E*****1 的大作中提到】
: Yes. It is true
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D*a
23
我是说你现在重新做以前的能不能看见带?

【在 E*****1 的大作中提到】
: Yes,I can before
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E*1
24


【在 D*a 的大作中提到】
: 我是说你现在重新做以前的能不能看见带?
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E*1
25
yea

【在 D*a 的大作中提到】
: 我是说你现在重新做以前的能不能看见带?
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E*1
26
The possibilty of gel:
Impossible, I tried gel from both pre-made of biorad and home-made
neither works
I checked TBS, PH is 7.6, a little bit high but not that high
I doubt maybe protein penetrate through PVDF membrane but like sunnyday
said "即使转膜不完全,不可能一点都看不到的"
It is so werid!
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g*5
27
Recently I used actin antibody (sigma) to probe mouse heart sample. no band
observed.
and actin can't recognize mouse muscle samples, too.
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s*y
28
Sigma 的哪个 actin 抗体啊? 如果是那个针对non-muscle actin 做出来的抗体,
在心脏和肌肉里没有信号难道不是很正常么?

band

【在 g*********5 的大作中提到】
: Recently I used actin antibody (sigma) to probe mouse heart sample. no band
: observed.
: and actin can't recognize mouse muscle samples, too.

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D*a
29
楼主上面回我不是说只有新提的样品看不见带

【在 s******y 的大作中提到】
: Sigma 的哪个 actin 抗体啊? 如果是那个针对non-muscle actin 做出来的抗体,
: 在心脏和肌肉里没有信号难道不是很正常么?
:
: band

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s*y
30
她很确定的说以前的样品也都看不见带么?
如果是的话,得看看那个一抗或者二抗里是不是不小心加入了奇怪的东西。

【在 D*a 的大作中提到】
: 楼主上面回我不是说只有新提的样品看不见带
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E*1
31
I did the experiment a month ago, good.
I used the sample to re-run, actin cannot appear
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s*y
32
哦,那还真的很奇怪。
会不会你在buffer 里不小心加进了sodium azide?
另外,也有可能是你们的一抗被细菌污染了然后全部失效了。。。虽然这种情况很少见

【在 E*****1 的大作中提到】
: I did the experiment a month ago, good.
: I used the sample to re-run, actin cannot appear

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E*1
33
first antibody I put sodium azide
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g*5
34
A5441. you are right.

【在 s******y 的大作中提到】
: Sigma 的哪个 actin 抗体啊? 如果是那个针对non-muscle actin 做出来的抗体,
: 在心脏和肌肉里没有信号难道不是很正常么?
:
: band

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s*y
35
不要加那个东西,
那个东西会抑制HRP
如果不小心加了,必须注意用普通的PBST or TBST 多洗几次(最少5次),
洗液里和二抗里切切不可再加sodium azide

【在 E*****1 的大作中提到】
: first antibody I put sodium azide
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l*1
36
LZ 两个月前 不上心没细看这的suuny 的WB 经验回帖了吧 啊
回复]
[ 9 ]
发信人: sunnyday (飞鱼), 信区: Biology
标 题: Re: 刚才老板问我,are you shaking plates?郁闷
发信站: BBS 未名空间站 (Mon Apr 2 20:52:04 2012, 美东)
你是不是刚进实验室?如果是的话,忍一阵子吧。
一般都要经过这个阶段的。我以前刚进实验室的时候我老板恨不得盯着我用pipet,
一开始也是有点不爽,但是后来还是从她那里学到不少小窍门,还是很有用的。
相反,我第一个博士后的老板是那种三不管的,把我往实验室一扔自生自灭那种,
虽然貌似自由,但是后来让我挣扎了很久,很多东西我是非常久之后才发现自己
做错的了。比方说那时候在第一个老板那里用的是牛奶,但是觉得一抗太贵,
我很自觉的把一抗存起来用,然后很傻很想当然的往里面加了点sodium azide,
结果western blot 的结果总是抽风,时而好时而不好,什么一抗二抗的浓度
我调了又调,死活查不出原因。后来过了很久,才发现是这个azide的问题。
如果老板对我多关心一点,而不是简单的让我自己去调一抗二抗的浓度,
我可能就少走很多弯路了。

http://www.mitbbs.com/article_t/Biology/31653045.html
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E*1
37
Thanks for all of you.It really gave me some hints.
I was just thinking if i did not put sodium azide, The 1st antibody in BSA
is easy to go bad. I want to keep for while to use it (one or two or three
month). so I did that
Also my previous lab all did the same. Never happened this problem before.
Thanks again. You all are really nice
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s*y
38
抗体没有那么容易坏的。
有些商业出售的抗体里面加有低浓度的sodium zaide, 那个是因为他们考虑到你后面
要把抗体稀释几千倍才用,所以无所谓。如果你在你的已经稀释的working stock 里
加sodium azide 那是不提倡的。你如果需要反复用抗体的话, 不如冷冻起来更好。

【在 E*****1 的大作中提到】
: Thanks for all of you.It really gave me some hints.
: I was just thinking if i did not put sodium azide, The 1st antibody in BSA
: is easy to go bad. I want to keep for while to use it (one or two or three
: month). so I did that
: Also my previous lab all did the same. Never happened this problem before.
: Thanks again. You all are really nice

avatar
l*1
39
LZ can try PD training within suunyday lab a good mentor in WB plus xxx,
Sure.

【在 E*****1 的大作中提到】
: Thanks for all of you.It really gave me some hints.
: I was just thinking if i did not put sodium azide, The 1st antibody in BSA
: is easy to go bad. I want to keep for while to use it (one or two or three
: month). so I did that
: Also my previous lab all did the same. Never happened this problem before.
: Thanks again. You all are really nice

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a*7
40
2ab放室温太久了失效了吧?
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W*7
41
一抗不要紧,只要二抗没有就行。。
我的一抗都是加很多azide的。。有的放四度能用一年多。。如果放-20冻融几次就不行
了。。。
所以具体怎么存放用过的antibody还真是看你用的频率和抗体本身的半衰期了,不试不
知道啊。。

【在 s******y 的大作中提到】
: 抗体没有那么容易坏的。
: 有些商业出售的抗体里面加有低浓度的sodium zaide, 那个是因为他们考虑到你后面
: 要把抗体稀释几千倍才用,所以无所谓。如果你在你的已经稀释的working stock 里
: 加sodium azide 那是不提倡的。你如果需要反复用抗体的话, 不如冷冻起来更好。

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E*1
42
Try new BSA without any sodium azmide, still not work!!
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s*y
43
太奇怪了,你有同时做其他抗体的对照么来确定二抗肯定没有问题么?
必须用同一次配出来的二抗,一半来做actin, 另外一半来做对照抗体。

【在 E*****1 的大作中提到】
: Try new BSA without any sodium azmide, still not work!!
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z*a
44
can you upload your picture?
too much protein?

【在 E*****1 的大作中提到】
: Try new BSA without any sodium azmide, still not work!!
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i*l
45
co我的一抗都是加 azide的。so far so good
as long as 2nd antibody and washing buffer do not contain NaN3

【在 W****7 的大作中提到】
: 一抗不要紧,只要二抗没有就行。。
: 我的一抗都是加很多azide的。。有的放四度能用一年多。。如果放-20冻融几次就不行
: 了。。。
: 所以具体怎么存放用过的antibody还真是看你用的频率和抗体本身的半衰期了,不试不
: 知道啊。。

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i*l
46
prep new sample
start over again may be the fastest way
i hate trouble shooting

【在 E*****1 的大作中提到】
: Try new BSA without any sodium azmide, still not work!!
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b*s
47
Ab is less likely to go wrong if you can have signals from other probings.
your sample prep might be the target of troubleshooting.
1) did you freeze and thaw your sample frequently? - if yes, your current
samples are no longer reliable. make aliquotes in the future, or boil
protein right after lysis.
2) must snap freeze and thaw on ice for all ampules
3) double check your lysis process and use new protein samples -
microtubules can be very hard to dissolve or remain soluble

【在 E*****1 的大作中提到】
: Try new BSA without any sodium azmide, still not work!!
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s*8
48
My WB has a lot, lot, lot.... of problems with different kinds of antibody.
我的问题是忽好忽坏的, 有的时候连p53也不出.
看了你的回帖, 我 有几个问题, sample in SDS loading dye 开始就煮好的, 用的
好好的,然后下次在煮, 跑胶, 就不出信号了, 虽然我染了膜, 挺好的条带。 多
久能重复用这个sample哪?

【在 b****s 的大作中提到】
: Ab is less likely to go wrong if you can have signals from other probings.
: your sample prep might be the target of troubleshooting.
: 1) did you freeze and thaw your sample frequently? - if yes, your current
: samples are no longer reliable. make aliquotes in the future, or boil
: protein right after lysis.
: 2) must snap freeze and thaw on ice for all ampules
: 3) double check your lysis process and use new protein samples -
: microtubules can be very hard to dissolve or remain soluble

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b*s
49
once proteins were brewed in SDS-PAGE buffer with DTT or bME, they can be
stored at -20C and no need to be boiled again.
personally i prefer boiling samples freshly prior to gel loading.. my
samples can be stable for 1-2 months with several freeze-thaw cycles.

.

【在 s******8 的大作中提到】
: My WB has a lot, lot, lot.... of problems with different kinds of antibody.
: 我的问题是忽好忽坏的, 有的时候连p53也不出.
: 看了你的回帖, 我 有几个问题, sample in SDS loading dye 开始就煮好的, 用的
: 好好的,然后下次在煮, 跑胶, 就不出信号了, 虽然我染了膜, 挺好的条带。 多
: 久能重复用这个sample哪?

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b*s
50
查查看你用的抗体的说明书。有可能你用的beta-actin只识别单一种属,譬如人。

【在 E*****1 的大作中提到】
: I cannot get beta-actin, other proteins is ok.
: I tried several actin antibodies, none works.
: I am thinking maybe semi-dry transfer too long, but 11-40KD marker still
: there.
: Whats happened?

avatar
z*a
51
pic
pic
show the pic of your WB
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E*1
52
I think something with membrane?
I bought pre-made membrane all the moluclar weight around 43-20 KD is
nothing, But I can get tubulin
Another question, except actin and GAPDH
what else I can use for control?
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s*y
53
Tubulin is good as internal control as well.
We sometimes also use vinculin
It is possible your membrane has problem and didn't retain the small
proteins.

【在 E*****1 的大作中提到】
: I think something with membrane?
: I bought pre-made membrane all the moluclar weight around 43-20 KD is
: nothing, But I can get tubulin
: Another question, except actin and GAPDH
: what else I can use for control?

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E*1
54
Yes.
But we r doing some cytoskeleton molecular,
so I do not want to use tublin as interbal control

【在 s******y 的大作中提到】
: Tubulin is good as internal control as well.
: We sometimes also use vinculin
: It is possible your membrane has problem and didn't retain the small
: proteins.

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s*y
55
You can use vinculin too

【在 E*****1 的大作中提到】
: Yes.
: But we r doing some cytoskeleton molecular,
: so I do not want to use tublin as interbal control

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E*1
56
we do not have that antibody
This is my film:

【在 s******y 的大作中提到】
: You can use vinculin too
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E*1
57
hi
can you see my attachment?
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s*y
58
既然你怀疑是膜的问题,何不找另外一个实验室借一小点膜来重新转一次你的
蛋白?干嘛要和自己过不去啊?

【在 E*****1 的大作中提到】
: we do not have that antibody
: This is my film:

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M*e
59
我有类似的经历:检测不到一个ATCC cell line 的 beta-actin。
Cell line: SW780 (human urothelial carcinoma).
I bought this cell line twice from ATCC, but I could not detect beta-actin
at all. The expression of beta-actin as shown by Western blot in this cell
line was reported in the literature.
I could detect GAPDH in this cell line.
I could use the same beta-actin antibody (Sigma) to detect beta-actin in
other human cell lines (urothelial and non-urothelial carcinoma) using the
same antibody (Sigma).
I don't think that one of steps of the whole WB process (including
antibodies, buffer, membrane, transfer, samples, etc) went wrong.
I still don't know why I couldn't detect beta-actin in this cell line, but I
didn't bother to figure it out.
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E*1
60
All the low molecular cannot be seen in my membrane, what happened??
I borrowed th new membrane
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T*r
61
Try nitrocellulose membrane o.2um other than o.45um thickness
Try to reduce your transfer time
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