Name | 101Bio WB Stripping Solution | ||||||
Cat. # | P5W3 How to pay with Also can buy from : | ||||||
Application | This product is for maximum removal of antibodies from a membrane and preserving the integrity of the antigen in 12-15 minutes . This product is for research use only. | ||||||
Product Size | 250 ml | ||||||
Description | 101Bio WB Stripping Solution (stripping buffer) is designed to rapidly strip antibodies bound to antigens on Western blotting membranes using chemiluminescent substrate.One of the major advantages of chemiluminescent detection is high sensitivity and the ability to strip reagents from a blot and re-probe the same blot.Re-probing Western blots are very useful for optimizing the blotting conditions such as testing different antibody dilution and membrane blocking conditions. The key to this process is to use conditions that cause the release of antibody from the antigen without causing a significant amount of antigen to be released from the membrane. During the stripping procedure, small amount of antigen is inevitably lost from the membrane. It is important to minimize this loss by stripping the antibody under gentle conditions. 101Bio WB stripping solution can achieve maximum removal of antibodies from a membrane and preserving the integrity of the antigen in 12-15 minutes without using harsh conditions such as boiling and treated with strong reducing agents.A blot can be stripped and re-probed multiple times to visualize multiple proteins or to optimize experimental conditions without the need for running multiple gels.
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Shipping / Storage | Ship and store at room temperature. | ||||||
Shelf Life | 24 months | ||||||
Manual (protocol) | 101Bio.com WB Stripping Solution | ||||||
Components |
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Name | Cat. # | Size | ||
---|---|---|---|---|
101Bio HRP Substrate Kit | P5W1 | 30 ml /30 ml | ||
101Bio Antibody Enhancer | P5W2 | 30 ml | ||
101Bio WB Stripping Solution | P5W3 | 250 ml | ||
101Bio Ponceau S Stain | P5W4 | 250 ml | ||
101Bio Peroxidase Suppressor | P5W5 | 30 ml | ||
101Bio High Efficiency Protein Precipitation kit | P5W6 | 30 ml /30 ml | ||
101Bio Red Blood Cell Lysis Buffer | P5W7 | 250 ml | ||
101Bio WB Blocking Solution | P5W8 | 250 ml | ||
101Bio Denaturing Protein Solubilization Reagent | P5W9 | 10 ml | ||
101Bio Non-Denatured Protein Solubilization Reagent | P5W10 | 10 ml | ||
101Bio Protein Solubilization Reagent for MS | P5W11 | 2 ml | ||
101Bio SDS-Remover | P5W12 | 10 ml | ||
101Bio Albumin Depletion Reagent for Plasma and Serum | P5W13 | 10 ml | ||
101Bio Protein A+G Agarose Beads | W0349 | 1 ml | ||
101Bio Protease Inhibitor Cocktail (100x) | W2200 | 1 ml | ||
101Bio Yeast Total Protein Extraction Kit for SDS-PAGE | Y203-50 | 50 rxn |
Exosome Isolation | Purity > 95% | |
Protein Extraction | 1 min total protein, 40 min membrane protein | |
3D Cell Culture Gel | 30% < mkt=""> | |
PCR Kits | 50% < mkt=""> | |
Beta-Hexosaminidase Activity Colorimetric Assay | Fast and sensitive, High-throughput | |
Endotoxin-Free Plasmid Kits | maxi, midi and mini-prep |
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可分为以下几个类群:(1)依赖DNA的DNA聚合酶;(2)依赖RNA的DNA聚合酶;(3)依赖DNA的RNA聚合酶;(4)依赖RNA的RNA聚合酶。前两者是DNA聚合酶,它使DNA复制链按模板顺序延长。如在原核生物中仅就大肠杆菌中已被发现的就有三种(分别简称为PolⅠ,PolⅡ和PolⅢ等);DNA聚合酶只能在有引物的基础上,即在DNA或RNA引物的3′-OH延伸,这DNA的合成方向记为5′→3′。换言之DNA聚合酶催化反应除底物(αNTP)外,还需要Mg2+ 、模板DNA和引物,迄今细胞内尚无发现可从单体起始DNA的合成。同样,上述(3)和(4)是催化RNA生物合成反应中最主要的RNA合成酶,它们以四种三磷酸核糖核苷(NTP)为底物,并需有DNA模板以及Mn2 及Mg2 的存在下,在前一个核苷酸3′-OH与下一个核苷酸的5′-P聚合形成3′,5′-磷酸二酯键,其新生链的方向也是5′→3′。RNA聚合酶也大量存在于原核和真核生物的细胞中。如大肠杆菌RNA聚合酶分子量4.8×105,由5条多肽链组成,分别命名为α,α,β,β′,和γ,全酶可用α2ββ′λ表示。真核生物RNA聚合酶分子大于5×105,由10~12个大小不等亚基组成。聚合酶除作为自然界生命活动中不可缺少的组分外,在实验室中大多用作生命科学研究的工具酶类之一。
一、内切酶作用体系:(自建立,试过,效果很好)
EcoRⅠTaKaRa消化DNA37℃3h
ddH2O11ul
H溶液2ul
EcoRⅠ1ul
DNA6ul
37℃孵育3h
二、亚硫酸氢盐修饰过程:(翻译稿)
1.用适合的内切酶消化大分子量的DNA(避免目的基因被切割),或者通过把DNA悬于蛋白酶k/SDS缓冲液,通过26gaugeneedle5次,37℃孵育30min(降低DNA大小,利于充分变性)
2.加2.2ul新鲜配制的3MNaOH到18ulDNA样品中,搅拌混匀,短暂离心。
3.37℃孵育15min。(有的资料介绍说可以42度20min)
4.90℃孵育2min(95度5nin也可),置于冰上,短暂离心。
5.加208ul饱和的Na2S2O5PH5.0(BDH)。(加7.6gNa2S2O5到15ml水,加464ul新鲜配制的10MNaOH)
6.加12ul10mM氢醌(55mg加水50ml氢醌),混匀并短暂离心
7.加200ul矿物油,防止水分蒸发,限制氧化。
8.55℃孵育4-16h(水浴)。
9.吸弃上层矿物油。
10.①加1mlPromega’sWizardDNAClean-upresin按照指导书进行脱盐处理。每个管与2.5ml注射器相连,(注意连接紧)用吸管吸到注射器,底加2ml的离心管;轻轻加压,不要弄破膜;②加2ml80%的异丙醇洗涤,离心10000rpm,30s,底换管;柱子换到新1.5ml尖EP管,弃注射器;③加50ul预热水(60-70℃)放2-3min;离心(柱子与离心管一起,10000rpm,30s,DNA到EP管,弃柱子;
11.加50ul水到minicolumn室温下静置5min
12.离心20s收积洗提液,弃掉minicolumn
13.加5.5ul3MNaOH然后37℃孵育15min
14.短暂离心
15.加1ul糖原(10mg/ml)
16.加33.3ul5MNH4OAcetatePH7.0
17.加330ul冷(-20℃)100%乙醇,充分混匀
18.-20℃过夜(或者-70℃30min)
19.4℃下14000rpm离心15min
20.弃上清,加70%乙醇洗涤,4℃下14000rpm离心15min。
21.重悬DNA于10ul水中,室温下静置~2h,间隔旋涡振荡
22.-20℃保存DNA
hotstar酶做PCR,或者做Q-PCR
产生这一现象的原因在于 DNA合成酶只能沿5'-3'的方向合成DNA 而DNA本身的两条链又是反向分布的 所以就造成了只有一条链合成可以连续地进行下去(以它为模板的子链生成方向正好是DNA聚合酶可以直接提供的) 而后随链要盘绕成回环 反扭过来 才能合成
再合成起始的时候 DNA聚合酶是需要一段RNA引物的 在原核生物中这一引物是由dnaQ(一种酶)在已解旋的单链5'端合成,真核生物中也有对应的酶 由于后随链的合成不连续 所以每个片段都要有引物 在DNA合成结束的时候 这些引物要被切除 因而留下缺口 这时又要特定的酶去填补缺口(比如 大肠杆菌中的DNA聚合酶I)可是填补序列和周围序列间会有缺刻 也就是说他们交界处的3'-5'磷酸二脂键是断开的 这时需要DNA连接酶发挥作用 将其连好
所以 后随链上的缺刻多 还真够DNA连接酶忙一阵的 前导链上只有一开始有RNA引物 因此 最后也基本只有这个地方会用到连接酶
目前我准备做甲基化PCR,由于经费问题,可能无法购买高价的试剂盒。看过甲基化PCR的原理后,在考虑经过修饰后,是否可以用RT-PCR的试剂盒(有现成的)来做甲基化PCR。请大牛指导!另求各位推荐性价比高的亚硫酸盐修饰试剂盒,谢谢!
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