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Amicon Ultra-15 Centrifugal Filter Unit with Ultracel-30 membrane

带有Ultracel-30膜的Amicon Ultra-15离心过滤器

Company: EMD Millipore
Catalog#: UFC903024
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Measurement of Transferrin- and Non-transferrin-bound Iron Uptake by Mouse Tissues
Author:
Date:
2016-09-05
[Abstract]  Iron in blood plasma is bound to its transport protein transferrin, which delivers iron to most tissues. In iron overload and certain pathological conditions, the carrying capacity of transferrin can become exceeded, giving rise to non-transferrin-bound iron, which is taken up preferentially by the liver, kidney, pancreas, and heart. The measurement of tissue transferrin- and non-transferrin-bound iron (TBI and NTBI, respectively) uptake in vivo can be achieved via intravenous administration of 59Fe-labeled TBI or NTBI followed by gamma counting of various organs. Here we describe a detailed protocol for the measurement of TBI and NTBI uptake by mouse tissues. [摘要]  血浆中的铁结合其转运蛋白转铁蛋白,其将铁递送至大多数组织。 在铁过载和某些病理状况下,转铁蛋白的携带能力可能超过,导致非转铁蛋白结合的铁,其优先被肝脏,肾脏,胰腺和心脏摄取。 分别测量组织转铁蛋白和非转铁蛋白结合的铁(分别为TBI和NTBI)在体内的摄取可以通过静脉内施用59 Fe标记的TBI或 NTBI,然后是各种器官的γ计数。 在这里我们描述了测量小鼠组织的TBI和NTBI摄取的详细协议。

In vitro Deneddylation Assay
Author:
Date:
2016-03-20
[Abstract]  Nedd8 is a small ubiquitin-like protein (9 kDa) covalently attached to a conserved lysine residue of a cullin protein which is part of cullin-RING ligases (CRLs). CRLs are major E3 ligases important for protein ubiquitination in the ubiquitin-proteasome pathway (UPP). The activity of CRLs is regulated by cycles of neddylation (CulA-N8, ~98 kDa) and deneddylation (CulA ~89 kDa). The COP9 signalosome (CSN) and Deneddylase A (DenA) are capable of cleaving the isopeptide bond between Nedd8 and CullinA. In contrast to the single protein DenA, CSN is an eight subunit multiprotein complex. Protein crude extracts of different Aspergillus nidulans csn deletion strains were mixed with recombinant CSN subunits expressed and purified from Escherichia coli (E. coli). Western ... [摘要]  Nedd8是共价连接到作为cullin-RING连接酶(CRL)的一部分的cullin蛋白的保守赖氨酸残基的小的遍在蛋白样蛋白(9kDa)。 CRL是对泛素 - 蛋白酶体途径(UPP)中的蛋白质泛素化重要的主要E3连接酶。 CRL的活性通过脱甲基化(CulA-N8,〜98kDa)和脱甲基化(CulA〜89kDa)的循环调节。 COP9信号体(CSN)和丁二酸酶A(DenA)能够切割Nedd8和CullinA之间的异肽键。与单一蛋白DenA相反,CSN是八亚基多蛋白复合物。将不同的构巢曲霉csn 缺失菌株的蛋白质粗提物与从大肠杆菌(大肠杆菌)表达和纯化的重组CSN亚基混合。使用抗CulA或抗Nedd8抗体的Western杂交实验可以显示Nedd化化合物与Denedd化CulA的比率。使用deneddylation测定,我们可以显示CsnE是在体外连接7-亚基预组装的CSN的最后一个亚基,然后CSN可以通过金属蛋白酶亚基CsnE执行cullin deneddylation。该测定法是一种快速且非昂贵的方法,其显现了用于脱蛋白的蛋白质的酶活性。它还可用于测试除去来自构巢曲霉(构巢曲霉)或其他生物体中的底物的翻译后修饰的其它藻肽的活性。

Monoclonal Antibody Purification (Nicotiana benthamiana Plants)
Author:
Date:
2014-01-20
[Abstract]  Plant-based expression systems provide an alternative biomanufacturing platform for recombinant proteins (Matoba et al., 2011). In particular, plant virus-based vectors can overexpress proteins within days in the leaf tissue of Nicotiana benthamiana (N. benthamiana). To overcome the issues of genetic instability and limited infectivity of recombinant viruses, Agrobacterium-mediated delivery of “deconstructed” virus vectors has become the mainstay for the production of large and/or multicomponent proteins, such as immunoglobulin (Ig)G monoclonal antibodies (mAbs). Here, we describe a method of producing human IgG mAbs in N. benthamiana using the tobamoviral replicon vector magnICON®. The vector can express up to a few hundred mg of a ... [摘要]  基于植物的表达系统为重组蛋白提供了替代的生物制造平台(Matoba等人,2011)。特别地,基于植物病毒的载体可以在烟草(Nicotiana benthamiana)(本塞姆氏烟草)的叶组织中天数内过表达蛋白质。为了克服重组病毒的遗传不稳定性和有限的感染性的问题,土壤杆菌介导的"解构的"病毒载体的递送已经成为生产大和/或多组分蛋白如免疫球蛋白的主要方法Ig)G单克隆抗体(mAb)。在这里,我们描述了在N中产生人IgG mAbs的方法。本生烟草使用烟草花叶病毒复制子载体magnICON 。载体在7天内可以表达高达几百mg的mAb/kg叶材料。显示了分别用于广泛中和的抗HIV和抗流感mAbs VRC01和CR6261的代表性病例(Hamorsky等人,2013)。叶组织匀浆,通过过滤和离心澄清提取物。 mAb通过快速蛋白质液相色谱(FPLC)使用蛋白A亲和力和Phenyl HP疏水界面树脂纯化。

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