Recombinant Human Parkin W403A Protein, CF Summary
Product Datasheets
Carrier Free
CF stands for Carrier Free (CF). We typically add Bovine Serum Albumin (BSA) as a carrier protein to our recombinant proteins.Adding a carrier protein enhances protein stability, increases shelf-life, and allows the recombinant protein to be stored at a more dilute concentration.The carrier free version does not contain BSA.
In general, we advise purchasing the recombinant protein with BSA for use in cell or tissue culture, or as an ELISA standard.In contrast, the carrier free protein is recommended for applications, in which the presence of BSA could interfere.
E3-162
| Formulation | X mg/ml (X μM) in 25 mM Tris pH 8.5, 200 mM NaCl, 0.03% Brij 35, 10% (v/v) Glycerol, 5 mM TCEP |
| Shipping | The product is shipped with dry ice or equivalent. Upon receipt, store it immediately at the temperature recommended below. |
| Stability & Storage: | Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
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Reconstitution Calculator
Background: Parkin
The E3 Ubiquitin ligase Parkin (encoded by the PARK2 gene) is an essential part of the cellular machinery that participates in the removal of damaged mitochondria. Mutations in PARK2 are known to cause a form of Parkinson"s disease known as autosomal recessive juvenile Parkinson"s disease (AR-JP), and the mechanisms by which defective Parkin ligase contributes to the dopaminergic cell death in this disease is an area of intense investigation.
Reported substrates for Parkin include BCL2, GPR37, MIRO1, MFN1, MFN2, TOMM20, USP30, and many others. Parkin (an RBR-class Ubiquitin ligase) structures have recently been reported by multiple groups, and reveal that the ligase is folded upon itself to produce an auto-inhibited state. The auto-inhibition is relieved by interactions with PINK1 kinase (which can phosphorylate both Parkin and Ubiquitin at serine residue number 65) and pS65 phospho-Ubiquitin by mechanisms that are under investigation.
In vitro, wild-type Parkin may be activated by treatment with recombinant PINK1, or addition of low concentrations of pS65-phosphoubiquitin. However, Parkin W403Ademonstrates in vitro activityin the absence of these activators. Parkin has been reported to generate poly-Ubiquitin chains in K6, K11, K48, and K63 linkages both in vitro and in vivo. This recombinant protein is untagged.
- Bingol, B. et al. (2014) Nature 510: 370
- Ordureau, A. et al. (2014) Mol. Cell 56: 360
- Riley, B.E. et al. (2013) Nat. Comm. 4: doi:10.1038/ncomms2982
- Saraff, S.A. et al. (2013) Nature 496: 372
- Spratt, D.E. et al. (2013) Nat. Comm. 4: doi:10.1038/ncomms2983
- Trempe, J.F. et al. (2013) Science 340: 1451
- Wauer T. et. al. (2015) Nature 524: 370
- Wauer T. & Komander, D. (2013) EMBO J 32: 2099
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1．目的
学会PCR操作的基本技术。
2．原理
是将待扩增的DNA模板加热变性，与其两侧互补的寡聚核苷酸引物复性，然后经过耐热的DNA聚合酶延伸。再进入下一轮变性—复性—延伸的循环，n次循环后DNA可被扩增（1+X）n倍。其中<25nt的引物退火温度Tm=2（A+T）+4（G+C）。
3．器材
旋涡混合器，微量移液取样器，移液器吸头，0.2mlPCR微量管，双面微量离心管架，PCR仪，台式离心机，琼脂糖凝胶电泳系统，水漂，恒温水浴。
4．试剂
5U/μlTaqDNA聚合酶，PCR缓冲液（10×，MgCl2free），25mMMgCl2，dNTP，引物，模板质粒pBS-CHI，无菌ddH2O。
5．实验准备
dNTP混合液（每种25mM），TAE电泳缓冲液，荧光染料，10´加样缓冲液，1.5ml离心管装入铝制饭盒（灭菌）、移液器吸头装入相应的吸头盒（灭菌）。合成的引物：Senseprimer5＇-GGATCCACAATGATGAGAGCC-３＇
Antisenseprimer５＇-GATATCATGGTGAGGTAGCTAGCTT-３＇
目的基因模板质粒：已经克隆在pBS载体上的1128bp几丁质酶（chitinase，CHI）基因。
待扩增的CHI片段长度：996bp。
6．操作步骤
（1）在0.2mlPCR微量离心管中配制50μl反应体系。（以下加样量供参考，括号内是最终需要量，实验时需参照Taq酶说明书计算）
ddH2O32μl
10×PCRbuffer（不含MgCl2）5μl
25mMMgCl23μl
2.5mmol/LdNTP4μl（每种dNTP终浓度0.2mM）
10μmol/LPrimer12μl（12.5~25pmoles）
10μmol/Lprimer22μl（12.5~25pmoles）
模板质粒1μl（1×10-3pmoles）
5U/μlTaq酶1μl（5U）
总体积50μl
（2）根据厂商的操作手册设置PCR仪的循环程序：
①94℃5min
②94℃1min
③54℃1min
④72℃1min50s
⑤goto②29times
⑥72℃10min
（3）PCR结束后，取10μl产物进行琼脂糖凝胶电泳。观察胶上是否有预计的主要产物带。
（4）清理桌面，撰写实验报告。
（5）PCR产物可以直接与T载体连接（见实验五），然后转化感受态细胞（见实验八）。
7．思考
（1）复性温度如何确定？
（2）为什么要在最后延伸10min？
（3）是否有非特异性扩增产物（或引物二聚体），如何才能消除？
更多资讯请关注于http://www.chinazglab.com/
(2)操作多份样品时，应先配制反应混合液并分装，减少操作，避免污染，同时增加反应的精确度。试剂的配置和分装应在装有紫外灯的超净工作台或负压工作台操作。
(3)移液器和吸头需专用。由于移液器极易受气溶胶或模板核酸的污染，移液器应尽可能使用可替换或可高压处理的，吸头尽可能使用带滤芯的吸头。
(4)防止操作人员污染，使用一次性手套，EP管与吸头应一次性使用，吸头不要长时间暴露于空气中，避免气溶胶的污染。
(5)加样或吸取模板核酸时要十分小心，吸样要缓慢，防止样品进入移液器内；吸样时尽量一次性完成，忌多次抽吸，以免交叉污染或产生气溶胶污染。
(6)EP管打开应先离心，将管壁及管盖上的液体甩至管底部。开管动作要轻，以防管内液体溅出。若不小心溅到手套或桌面上，应立刻更换手套并用稀酸擦拭桌面。
(7)模板核酸应密封保存，防止外溢及外来核酸的进入。
(8)设立适当的阴阳性对照及重复实验，既可验证LAMP反应的准确性，又可以协助判断扩增系统的可信性。
(9)扩增产物应妥善处理，尽量避免开盖检测，开盖极易产生气溶胶污染。本公司的试剂均采用闭管检测，有效防止污染发生。展开
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