{{'Search' | translate}}
 

Lysozyme from chicken egg white

Lysozyme from chicken egg white

Company: Sigma-Aldrich
Catalog#: L6876
Bio-protocol()
Company-protocol()
Other protocol()

Purification of Soluble Recombinant Human Tau Protein from Bacteria Using Double-tag Affinity Purification
Author:
Date:
2018-11-20
[Abstract]  Dysfunction of the microtubule-associated protein Tau (encoded by the MAPT gene) has been implicated in more than twenty neurodegenerative diseases, including Alzheimer’s. As such, the physiological and disease-relevant functions of Tau have garnered great interest in the research community. One barrier hampering investigations into the functions of Tau and the generation of pharmacological agents targeting Tau has been the difficulty of obtaining soluble Tau protein in purified form. Here, we describe a protocol that uses dual affinity tag purification to selectively purify soluble recombinant Tau protein from bacteria that is functionally active for downstream applications including immunization, microtubule binding assays, and protein-protein interaction studies. [摘要]  微管相关蛋白Tau(由 MAPT 基因编码)的功能障碍已经涉及20多种神经退行性疾病,包括阿尔茨海默病。 因此,Tau的生理和疾病相关功能引起了研究界的极大兴趣。 妨碍对Tau功能的研究和产生靶向Tau的药理学试剂的一个障碍是难以获得纯化形式的可溶性Tau蛋白。 在这里,我们描述了一种方案,该方案使用双亲和标签纯化从细菌中选择性纯化可溶性重组Tau蛋白,所述细菌对于下游应用具有功能活性,包括免疫,微管结合测定和蛋白质 - 蛋白质相互作用研究。
【背景】Tau传统上被定义为微管结合蛋白;然而,在人类疾病中,Tau可以与轴突微管分离并错误定位到其他神经元区室,包括体细胞,树突和突触,其中与非微管蛋白和结构的相互作用驱动神经元功能障碍(Iqbal et al。 ,2016; Wang和Mandelkow,2016; Zhou et al。,2017; McInnes et al。,2018)。尽管神经原纤维缠结形式的Tau聚集体通常存在于死后患病的脑组织中,但研究表明,可溶性Tau,而不是聚集的Tau,是神经元功能障碍的主要原因(Crimins et al。,2012 ; Polydoro et al。,2014; Koss et al。,2016)。因此,研究Tau在疾病中的可溶性功能,例如鉴定蛋白质 - ...

Identifying Protein Interactions with Histone Peptides Using Bio-layer Interferometry
Author:
Date:
2018-09-20
[Abstract]  Histone post-translational modifications (PTMs) regulate numerous cellular processes, including gene transcription, cell division, and DNA damage repair. Most histone PTMs affect the recruitment or exclusion of reader proteins from chromatin. Here, we present a protocol to measure affinity and interaction kinetics between histone peptides and the recombinant protein using Bio-layer interferometry. [摘要]  组蛋白翻译后修饰(PTM)调节许多细胞过程,包括基因转录,细胞分裂和DNA损伤修复。 大多数组蛋白PTM影响从染色质中募集或排除读取蛋白。 在这里,我们提出了一个协议,使用生物层干涉测量法测量组蛋白肽和重组蛋白之间的亲和力和相互作用动力学。

【背景】真核染色质结构大致分为常染色质和异染色质(Cheung和Lau,2005),异染色质结构根据组蛋白翻译后修饰(PTM)的组合进一步细分。这些PTM不仅改变染色质构象,还在基因表达和蛋白质募集中建立直接调节作用(Felsenfeld和Groudine,2003; Allshire和Madhani,2017)。组蛋白PTM的无数组合 - 包括乙酰化,磷酸化,甲基化,泛素化,生物素化,SUMO化和脯氨酸异构化,统称为“组蛋白标记” - 可以被发现,特别是在从核小体核心突出的非结构化N末端尾部( Guetg和Santoro,2012)。这些PTM通过不同“读者”或效应蛋白的活动调节许多细胞过程,包括基因转录,细胞分裂和DNA损伤修复(Suganuma和Workman,2011)(Musselman et al。, 2012)。因此,已经做出很大努力来识别读者的组蛋白修饰。

使用常规方法(例如,表面等离子体共振[SPR]和SPR成像[SPRi]生物传感器)研究读取蛋白与其靶蛋白PTM之间的相互作用通常需要大量底物或复杂的多步实验方法并且由于各种方法特定的限制而变得复杂。这些问题排除了量化相互作用强度的简便性和准确性(Phizicky和Fields,1995; ...

In vitro Chaperone Activity Assay Using α-Amylase as Target Protein
Author:
Date:
2018-06-20
[Abstract]  Small heat shock proteins (sHSP) are stress proteins which are ubiquitously found in almost all living organisms. They function as molecular chaperones, which assist in protein folding during translation and in the prevention of irreversible protein aggregation under denaturing conditions. This protocol describes the use of α-amylase as target protein in assessing the chaperone activity of wild and mutant recombinant small heat shock proteins of Mycobacterium leprae. Chaperone activity of these proteins, along with α-crystallin, a standard sHSP was demonstrated using a new method employing their protective effect against heat denaturation of α-amylase from porcine pancreas. The regained enzymatic activity of the α-amylase was demonstrated on starch agar plates stained with ... [摘要]  小热休克蛋白(sHSP)是在几乎所有生物体中无处不在发现的应激蛋白。 它们作为分子伴侣起作用,这有助于在翻译过程中蛋白质折叠以及在变性条件下预防不可逆的蛋白质聚集。 该协议描述了使用α-淀粉酶作为靶蛋白来评估麻风分枝杆菌的野生和突变重组小热休克蛋白的分子伴侣活性。 这些蛋白质的陪伴分子活性以及标准sHSP的α-晶状体蛋白通过采用其对猪胰α-淀粉酶的热变性的保护作用的新方法被证实。 在用碘 - 碘化钾(I 2 -KI)溶液染色的淀粉琼脂平板上证实α-淀粉酶的重新酶活性。

【背景】热休克蛋白(HSPs)是一组保守的蛋白质,当细胞暴露于外部应激(包括热应激和冷应激)时诱导蛋白质。该组中的大多数成员在功能上与蛋白质折叠和解折叠机制有关。小热休克蛋白(sHSPs)是热休克蛋白的子集,其分子大小为12至43kDa,并且保守的C末端区域称为'α-晶域'。 sHSP通过与部分未折叠的蛋白结合并阻止其完全变性而显示ATP非依赖性分子伴侣活性。有几种用于证明sHSPs的体外伴侣蛋白活性的方法,其使用各种底物蛋白如RuBisCO(Goloubinoff等人,1989),rhodanese(Mendoza等人(Farahbakhsh等,1995),溶菌酶(Rozema和Gellman,1996),苹果酸脱氢酶(Lee等, ...

Comments