m*********7
发帖数: 606 | 1 第一，你的tissue lysate测了总蛋白浓度没有？
第二，如楼上所说，你既然要做Western blot，为什么不用Western blot的常规方法用抗体去检测持家基因的表达量？
第三，谁能告诉我那些状如小气泡的东西是什么啊？难道是你的胶上就含着这些气泡？
第四，为什么这块胶泡了１０个月你才来问上样量可不可比？
第五，你为什么会认为别人能够目测蛋白浓度，就凭这张照片？
第六，５００ng蛋白接近commassie blue染色检测的下限了（有文献说下限为0.3-1ug/lane，与染色条件相关），你看不见也属正常。 |
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S******e
发帖数: 393 | 2 传统方法，1 X transfer buffer: 30mM Tris, 240mM Glycine, 20% methanol，
另外可加可不加SDS(0.2%)。
我一般40伏过夜，nitrocellulose membrane。
另外，也不一定是western问题，首先还是要确定你的基因/蛋白是不是表达了，
做个瞬转吧，250kD 不太难，我做过这么大蛋白瞬转的，做western也检测到了表达。 |
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A*******e
发帖数: 284 | 3 请教，我用GFP融合一个转录因子表达，用western测表达量。现在发现表达量不高，
所以western结果不漂亮，free GFP的control倒是浓的一塌糊涂，请问有什么可以改进
的？
我用santa cruz的GFP-HRP抗体，现在看来似乎不好，出现了若干杂带，原本表达量就
不高，还搞些杂带就更麻烦了。 可有好的偶联HRP或AP的GFP抗体推荐， 多谢了 |
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A*******e
发帖数: 284 | 4 多谢各位的指点。我这个protein是个TF，差不多是个全新的蛋白，所以没有specific
antibody. 我加GFP是因为我想看它的subcellular localization. 如我前面说的，我
的vector没有问题，因为free gfp很亮，无论是看荧光还是western。 我的融合蛋白是
我把我的protein放N段，中间一点link， 还有一段his， 然后就是GFP。 在荧光下我
能看得到特异定位于核的GFP信号，所以我觉得我的construct没有问题。 但是western
出来的带就是非常弱，尤其是我把free gfp vector放一边做control的时候。 我觉得
问题是蛋白表达量很低，我已经把上样量放到极限了。 上面各位给的很好的建议，我
想都试试看 |
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g***s
发帖数: 30 | 5 我们的方法是：裂解buffer：RIPA ，裂解方法：磁珠快速震荡，破碎larva。然后离心
，加入SDS loading buffer.
结果western结果很奇怪，经常出现一部分样品western没有任何信号，另外一部分完全
正常的情况。跟抗体肯定没关系，因为我们用一些非常好用的如tubulin actin都试过
了。
各位有遇到过类似的问题吗？ 能给支支招吗 |
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l******n
发帖数: 298 | 6 小老板最近做了一个tissue specific expression of AKT mice.上面还连了一个HA.免
疫染色跟wt比较能看出挺大差别。但是我提了各个组织，用western 检测不出来是怎么
回事呢？
我用kit 提的总蛋白，取了肝啊啥的几个器官，western上样100ug/lane.应该足够了。
另外谁能推荐一个好的HA 抗体，我用了sigma H9658和roche 12CA5,感觉都一般。
多谢！ |
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l**********1
发帖数: 5204 | 7 按巴黎统筹规定
禁止出口后清的吧
全自动的western blot machine Made in Germany/France/USA
//www.referenceforbusiness.com/encyclopedia/Con-Cos/Coordinating-Committee-for-Multilateral-
Export-Controls-and-the-Wassenaar-Arrangement.html#b
証明和军用单位完全无关以前 比如美国总领事馆以内用的除外
http://www.mitbbs.com/article_t/Biology/31590799.html
同主题阅读：有人用过或者知道自动的western blot machine 么？
[版面:生物学][首篇作者：sunnyday] , 2011年10月24日 |
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c**a
发帖数: 94 | 8 384的盘子用CHO做ICW, 种细胞之后和第二天早上都好好的, 到了做serum starve 之后
一看好多细胞被洗掉了,但是奇怪的是正中间的不掉,贴壁一圈不掉,而是周边的细胞都
没了, western染色之后看起来像个'回'字. 具体样子在附件里. 我确定的一点是之后
的treatment 和western步骤并没有显著的让细胞减少, 所以貌似问题在用plain
medium 洗细胞和serum starvation步骤上. 是这样的吗, 怎么避免呢, 洗的时候很
gentle了, 怎么可以更gentle呢? |
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l****n
发帖数: 58 | 9 近期投的一篇稿件。
Statistical reviewer认为western和qPCR样本小, quantification的图应该用 dot
diagram 而不是 bar graph。
我翻遍了文献看到的western和qPCR都是用bar graph啊。该怎么回他啊。谢谢了! |
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l****n
发帖数: 58 | 10 说实话20%左右的变化我也是不信的，western个人感觉要肉眼就能看到差别才算真有改
变。很多文章其实western都不做quantification。 |
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l**********1
发帖数: 5204 | 11 sure
LZ 考古下
Re: 有人做过10kDa蛋白的western嘛？
[版面:生物学][首篇作者：cheesecake] , 2006年09月21日12:39:58
[分页:1 ]
[ 1 ]
发信人: cheesecake (cake), 信区: Biology
标 题: Re: 有人做过10kDa蛋白的western嘛？
发信站: BBS 未名空间站 (Thu Sep 21 12:39:58 2006)
I agree with that.
I used 4-15% geadient and 0.45um membrane (.2 is better) and tested 7KD
protein without any problem.
http://www.mitbbs.com/article_t/Biology/11565847.html |
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d****i
发帖数: 2346 | 12 我们使用的是荧光二抗，就是licor的那个，不是HRP，做某蛋白的磷酸化分析，
western。
问题一，一般是先上磷酸化抗体，strip之后再上total抗体，但是这种二抗strip掉信
号很困难，大家有什么好办法？
问题二，western里有磷酸化抗体的信号，total蛋白的信号，还有actin的信号，怎么
计算磷酸化信号的强弱？
谢谢！ |
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e*******c
发帖数: 1479 | 13 老板忽发奇想， 不用血浆/血清做ELISA, 要我为他做western， 做了一边，直接将血
浆变性上样， 跑SDS-PAGE. western在50多的位置太多的蛋白模糊不清， 有无作过的
同学给给经验
谢谢 |
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M*******C
发帖数: 183 | 14 以前我们用ECF底物，strip用0.2N NaOH 就好了。
现在ECF 买不着了，改用ECL plus，Thermo Fisher/Pierce （32132）的，发现用NaOH
没法strip了，第一次blot的带仍然在那里。 看了ECLplus 的说明书，他们叫用
Thermo Scientific™Restore™ Western Blot Stripping Buffer (
Product No. 21059) 或者Restore Plus Western Blot Stripping Buffer (Product
No. 46430). ， 价格 500ml/$130, 太贵了，strip 一张膜得10ml 吧，500ml 50张膜。
不知道成分是什么，想自己配点，求各位指点！谢谢 |
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r***r
发帖数: 22 | 15 【 以下文字转载自 Oversea 讨论区 】
【 原文由 raner 所发表 】
要在HK买点东西, 只能bank transferation或Western Union两种.
不知道有清楚这两种方式的网友吗? fee? speed?
查了一下, local还有些western union的agent. bank transfer能把
money直接转到Asia的bank里吗? |
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a********e
发帖数: 3771 | 16 【 以下文字转载自 Biology 讨论区 】
发信人: andyzzdlee (麦地之一看二慢三通过), 信区: Biology
标 题: western提蛋白的困惑：多次离心vortex会不会降解蛋白
发信站: BBS 未名空间站 (Wed Jan 20 23:38:17 2010, 美东)
最近一次做western，用NEPER kit提取胞浆里的ERK和p38蛋白。因为用划伤组织做刺激
，脱落不少细胞，我就把medium离心得到这些脱落的细胞。同时在培养皿里加冰PBS，
在冰PBS里刮下剩余的细胞放进脱落的细胞里再次离心。然后用kit里CER lysis buffer
裂解（已加入inhibitor cocktail）。
裂解的时候我vortex两次，每次15sec。NEPER protocol要求vortext一次，15sec即可。
结果照片洗出来什么都没有，连EGF刺激的positive control都没出来。
以前只用过一次NEPER，erk出来band，而且结果可信。不同点在于没有收集脱落的细胞。
所以我考虑是不是多次离心、vortex是蛋白降解，也可能是收集脱落细 |
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J**********i
发帖数: 925 | 17 Chinese Medicine, Western Style
Jim Kling
United States
8 December 2006
China’s economy is the third largest in the world behind the U.S. and the
European Union, according to the World Fact Book, and it continues to grow
at a rapid rate. What does that mean for science careers? It’s a muddled
picture, as opportunities in different fields grow at vastly different rates
depending on social and economic factors.
But at least one western industry--pharmaceuticals--is positioning itself to
be a part |
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b*****9
发帖数: 8922 | 18 ☆─────────────────────────────────────☆
GeorgeCobain (铁丝钢琴手) 于 (Wed Sep 9 04:26:22 2009, 美东) 提到:
China's Western Front
Can Beijing Bring Order to Its Restive Provinces?
Christian Le Mière
CHRISTIAN LE MIÈRE is Editor of Jane's Intelligence Review.
Early last month, the mayor of Urumqi, the capital of the western Chinese
province of Xinjiang, described the struggle to maintain China's unity as "a
political battle that's fierce and of blood and fire." His description was
apt: a spate |
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a***a
发帖数: 12425 | 19 今天打到的，新鲜出炉。前三张是一只鸟，我脚得就是Western Bluebird，属于亮肤色
；后四张是另一只，我不是很确定，但是看到鸟书上的图(P401)，脚得很像那种drab
adult（土色？）的此类鸟。。或者（1）-（3）是雄鸟、（4）-（7）是雌鸟？谁给指
点指点。。
（1）-（3)是因为我坐在那里然后他在落下来，比较近，我赶紧拍，所以比较清晰，看
来距离是关键啊。
冰总贴过Estern Bluebird，对照看能获得更多惊喜呦～
http://www.mitbbs.com/article_t/Animals/76137.html
http://www.mitbbs.com/article_t/Animals/81613.html
==========================================
补充：在冰总的帮助下，注意到“鸟书上Drab Adult的旁边有个雌性的符号，应该是说
只有雌性才会可能有这种颜色。”多谢李冰冰。那么（1）-（3）是雄鸟，（4）-（7）
是雌鸟，我在同一天都拍到啦！
===============================... 阅读全帖 |
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a***a
发帖数: 12425 | 20 前几天去一个地方走了走，途中眼前突然一道五彩霞光闪过，然后光停在了灌木丛中。
我定睛一瞧，哎呦喂，一只从没见过的颜色艳丽的小鸟！我赶紧拿相机，拍照，可惜鸟
儿发觉了我，我只拍了两张他就飞走了。后来又在路上看到同一种类的鸟儿在半空飞翔
，这鸟儿颜色靓丽，十分打眼儿。回来看了看，照片虚了，但还是要放上来。
我每次打到新鸟种最大的难题就是认鸟儿，本来脚得这次这个又是个难题。刚巧儿，鸟
书扣着放，我一看，哎呦喂，不就是他吗～哈哈哈，介鸟儿出现在鸟书的封底，一模一
样啊，原来叫Western Tanager（正文P463）。我见到的这只是在breeding阶段的雄性
。这种鸟多数分布在西南，鸟书封底鸟儿的另一只叫Scarlet Tanager，多数分布在东
南，东部的童鞋可以多踅摸踅摸，呵呵。
（1）
（2）
Western Tanager肖像图。 |
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J*****s
发帖数: 185 | 21 http://popwatch.ew.com/2010/06/30/rango-johnny-depp/
by John Young
Categories: Animation, Movie Trailers, Movies, On the Scene
Image Credit: Paramount PicturesThe first proper trailer for the CG-animated
film Rango was unleashed yesterday (and embedded below), and despite its
peyote-infused imagery, it made a hell of a lot more sense than the wind-up
goldfish teaser that popped up a few weeks ago. But what if you wanted a bit
more dirt about this Gore Verbinski-directed, Johnny Depp-starring liz... 阅读全帖 |
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w*******y
发帖数: 60932 | 24 Best Buy has Western Digital - Scorpio Blue 320GB 2.5" Internal Serial ATA
Hard Drive for Laptops (OEM/Bare Drive)
$34.99 + Ship
Link:
http://www.bestbuy.com/site/Western Digital - Scorpio Blue 320GB Internal Serial ATA Hard Drive for Laptops (OEM/Bare Drive)/9861731.p?skuId=9861731&productCategoryId=pcmcat189600050008&id=1218186269455
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w*******y
发帖数: 60932 | 25 Best Buy has the Western Digital - Scorpio Blue 320GB Internal Serial ATA
Hard Drive for Laptops (OEM/Bare Drive) for $35 with free shipping.
Link:
http://www.bestbuy.com/site/Western Digital - Scorpio Blue 320GB Internal Serial ATA Hard Drive for Laptops (OEM/Bare Drive)/9861731.p?id=1218186269455&skuId=9861731
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w*******y
发帖数: 60932 | 27 Western Digital Elements 2TB USB 2.0 Desktop External Hard Drive
WDBAAU0020HBK-NESN $109.99 Shipped
Western Digital Elements 2TB USB 2.0 Desktop External Hard Drive
WDBAAU0020HBK-NESN:
ends 8/7
|
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w*******y
发帖数: 60932 | 28 Newegg.com has the Western Digital WD TV Live HD Media Player (Recertified)
for $59.99 plus $4.99 shipping.
Link:
http://www.newegg.com/Product/Product.aspx?Item=N82E16822136628
Comes with 180 Day Warranty.
Model
Brand Western Digital
Model WDBAAN0000NBK
Performance
Interface USB 2.0
Physical Spec
Dimensions 4.94" x 3.94" x 1.57"
Weight 0.67 lbs.
Features
Features Full-HD video playback and navigation - up to 1080p
Play a wide variety of file formats
Play videos, music an |
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w*******y
发帖数: 60932 | 29 Amazon:
http://www.amazon.com/Western-Digital-Caviar-Desktop-WD1002FAEX/dp/tech-data/B0036Q7MV0/
has the Western Digital Caviar Black 1 TB Desktop Hard Drive (WD1002FAEX)
on sale today only for $76.49 - 10% off code GXTPOFES (only works if you
have not used the code before) = $68.84 with free super saver shipping. Next
lowest on Google Products is $85.
Product Features
* WD Caviar Black high performance 3.5- Inch SATA hard drive combines
7200 RPM, 64 MB cache, and SATA 6 Gb/s interface for |
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w*******y
发帖数: 60932 | 30 Newegg's:
http://www.newegg.com/Product/Product.aspx?Item=N82E16822136296
Shell Shocker deal has the Western Digital VelociRaptor WD1500HLFS 150GB
10000 RPM SATA 3.0Gb/s 3.5" Internal Hard Drive -Bare Drive for $99.99
shipped. That's 29% off it's normal $139.99 price.
Deal is over at 1:00pm (PDT).
Model
Brand Western Digital
Series VelociRaptor
Model WD1500HLFS
Packaging Bare Drive
Performance
Interface SATA 3.0Gb/s
Capacity 150GB
RPM 10000 RPM
Cache 16MB
Average Seek |
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w*******y
发帖数: 60932 | 32 Newegg:
http://www.newegg.com/Product/ComboDealDetails.aspx?ItemList=Combo.493043
has a combo deal: Western Digital WD20EADS 2TB HD and LG 22X DVD Burner
combo for $109 with free shipping. This is a "Shell Shocker" deal and
expires @ midnight PST.
1. Western Digital Caviar Green WD20EADS 2TB SATA 3.0Gb/s 3.5" Internal Hard
Drive -Bare Drive
2. LG 22X DVD Burner - Bulk Black SATA Model GH22NS40 - OEM
Both items are normally $147.98
Combo Discount: $38.98
Today's Deal Price: $109.00
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w*******y
发帖数: 60932 | 33 Newegg has the Western Digital VelociRaptor WD1500HLFS 150GB 10000 RPM SATA
3.0Gb/s 3.5" Internal Hard Drive -Bare Drive for $99.99 shipped. That's 29%
off it's normal $139.99 price.
Coupon code: EMCYXYN36
Model
Brand Western Digital
Series VelociRaptor
Model WD1500HLFS
Packaging Bare Drive
Performance
Interface SATA 3.0Gb/s
Capacity 150GB
RPM 10000 RPM
Cache 16MB
Average Seek Time 4.2ms
Average Write Time 4.7ms
Average Latency 3ms (nominal)
Physical Spec
Form Fa |
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w*******y
发帖数: 60932 | 35 Amazon:
http://www.amazon.com/Western-Digital-Caviar-Desktop-WD2001FASS/dp/B002MD05SA/
has the Western Digital 2 TB Caviar Black SATA 7200 RPM 64 MB Cache Bulk/
OEM Desktop Hard Drive WD2001FASS on sale today only for $139.99
Product Features
* WD Caviar Black high performance 3.5-inch SATA hard drives combine
7200 RPM spin speed, 32 MB or 64 MB cache
* High performance electronics architecture features dual processors and
bigger
* StableTrac - The motor shaft is secured at both en |
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w*******y
发帖数: 60932 | 38 Smartbargain s.com
They are tricky with links, but some nice deals on brand rain boots. I have
never priced rain boots before I ran a Target thread about a week back, and
there was a bunch of people cancelled on Hoping this helps them-there are
some pretty nice styles for the $25 bucks. Hard to price shop these as they
arent widely available. But Victoria Safari and Leopard seems pretty pricey
(90+) elsewhere.
You need to be careful with links. Shop from the first link. Keep that
browser open, ... 阅读全帖 |
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w*******y
发帖数: 60932 | 40 The Western Digital - My Book Essential 1.5TB External USB 2.0 Hard Drive
will be $79.99 at Best Buy 11/21-11/24
Link:
http://www.bestbuy.com/site/Western Digital - My Book Essential 1.5TB External USB 2.0 Hard Drive/9467196.p?id=1218109896939&skuId=9467196&st=9467196&cp=1&lp=1
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w*******y
发帖数: 60932 | 41 Hey everyone,
Amazon will have a portable 500 GB Western Digital 500 GB for "under $40" at
4:45 AM PST, which means it'll probably be $39.99. Chances are fair that
this is the Link:
http://www.amazon.com/Western-Digital-Elements-Portable-WDBAAR5000ABK-NESN/dp/B002CZQ4GU/
, which is currently selling for $55.
Pretty good price if you're in the market for a 500 GB portable.
Remember, the deal comes live in an hour and 10 minutes, so get ready --
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w*******y
发帖数: 60932 | 47 LINK:
http://www.newegg.com/Product/Product.aspx?Item=N82E16822136767&Tpk=Western Digital Caviar Blue WD10EALX 1TB 7200 RPM
With Promo Code EMCZNNT35
Cache:32MB
Features:Solid performance - WD Caviar Blue drives are built to deliver rock
-solid performance ideal for family and business computing. Ultra cool and
quiet - Keeping the drive cool enhances reliability. WD continues to develop
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helps protect the
Parts:3 years lim... 阅读全帖 |
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w*******y
发帖数: 60932 | 50 * Join Best Western Rewards our FREE rewards program, and set your earning
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