植物组织中蛋白质及同功酶的聚丙烯酰胺凝胶盘状电泳

聚丙烯酰胺凝胶电泳是鉴定蛋白质、酶的有力工具,是生物化学研究上不可缺少的实验技术。聚丙烯酰胺凝胶是一种合成的凝胶,是由丙烯酰胺单体和交联剂甲义丙烯酰胺在催化剂的作用下聚合而成的大分子。不同的甲义丙烯酰胺在和丙烯酰胺浓度可制成孔径不同大小的电泳基质。电泳时蛋白质混     

电泳用水平聚丙烯酰胺干胶研制成功

聚丙烯酰胺凝胶电泳是快速低廉的生化分析技术。由于凝胶具有三维网状结构,电泳时兼具有分子筛效应和电荷效应,从而比其他电泳的分辨力大为提高,并在此基础上又发展了ＳＤＳ聚丙烯酰胺电泳、梯度胶电泳和等电聚焦聚丙烯酰胺电泳等技术。除大量用于各种蛋白质分析、分型...     

一种检测聚丙烯酰胺凝胶中蛋白质免疫活性的方法

聚丙烯酰胺凝胶电泳(PAGE)已成为最有效的分离蛋白质手段。在免疫学研究中,经常遇到的问题是从聚丙烯酰胺凝胶中回收被分开的蛋白质组分,然后测其免疫活性。从聚丙烯酰胺凝胶中回收蛋白质的方法已有许多报道,其中以Hartvig的方法为最简便。该法以溴酚蓝为指示剂,利用电泳系统本身使     

化工上的应用

<正> 853780 蛋白质分馏过程中的考马斯亮蓝 R-250聚丙烯酰胺着色凝胶[英]/Borejdo,J.…∥Anal.Biochem.-1984,140(1).-84～86[译自 DBA,1984,3(20),84-09885]介绍一种使聚丙烯酰胺凝胶染色的方法,这种方法使染料和试样一起进行电泳。     

意蜂与中蜂血淋巴蛋白质成份的研究

本实验用聚丙烯酰胺凝胶电泳分析了两个蜂种的血淋巴蛋白质成分。意蜂和中蜂都是Apis属,血淋巴蛋白质电泳谱相似。但是,两蜂种以及同一蜂种不同级别、不同发育阶段的电泳谱又各有特点。根据实验,我们把成年蜂血淋巴电泳谱分为11条带,雌蜂电泳谱上的带5是卵黄原蛋白;雄蜂卵黄原蛋白含量很少或测不出。 用Thorun方法,在聚丙烯酰胺凝胶平板电泳上测得意蜂卵黄原蛋白的分子量为185,000。     

用有限酶切法分析蛋白质的氨基酸序列

报道了一个通过有限酶切蛋白质产生多肽片段的方法.蛋白质经单向SDS-聚丙烯酰胺凝胶电泳(SDS-PAGE)分离和用考马斯亮蓝短暂染色后,切下所需的蛋白质带,将其放入另一个SDS-PAGE凝胶的样品槽内,在电泳过程中该蛋白质被蛋白酶如蛋白酶V8降解,所产生的多肽片段随之被分离.电泳结束后,将多肽片段电印迹至聚偏二氟乙烯(polyvinylidene difluoride,PVDF)膜上.这些多肽片段从PVDF膜上切下后可以直接被用于分析氨基酸序列.该方法能广泛适用于分析一般蛋白质和N端被修饰蛋白质的氨基酸序列.     

一种提高固定pH梯度（IPG）凝胶双向电泳的重复性、分辨率和通量的方法

在蛋白质组学研究中 ,双向聚丙烯酰胺凝胶电泳是现行蛋白质分离的最重要的方法之一。实验发展了一种提高固定pH梯度 (IPG)凝胶双向电泳的重复性、分辨率和通量的方法 :在一块SDS 聚丙烯酰胺凝胶上同时进行多块固定pH梯度(IPG)凝胶 (Multi stripsononeSDSgel,MSOG)电泳。用此方法比较了人肝癌细胞、不同生长状态的人肝癌细胞、3T3细胞的蛋白质以及同一个样品在不同大小的第二向凝胶系统 (大型和中型凝胶 )的双向电泳图谱。结果表明 ,同一样品在 13cmIPGStrip双向电泳可分离 2 0 0 0以上蛋白质点且图谱蛋白质点的匹配率可超过 95 %以上。同时又可以最大程度地降低凝胶背景对蛋白质点比较分析的干扰 ,从而提高了双向电泳分离蛋白质的分辨率和通量。这些优点都有助于差异蛋白质组学特别是细胞器差异蛋白质组学研究的自动化。     

辽宁产东亚钳蝎(Buthus martensii Karshi)毒两种毒素的纯化及其部分性质的研究

用CM-Sephadex C-50分离辽宁产东亚钳蝎毒得到十二个蛋白组份,对其中的第八组份进行了CM-Sephadex C-50重层析和Sephadex G-50凝胶过滤,最后得到两种纯化的毒素。应用低pH系统不连续聚丙烯酰胺凝胶圆盘电泳、SDS-不连续聚丙烯酰胺凝胶板状电泳及等电聚焦聚丙烯酰胺凝胶圆盘电泳鉴定均为单一条带。二者的分子量和等电点分别为8,980,8,660和7.58,7.90。还测定了粗毒对小白鼠的LD50(腹腔注射)、有关酶活力和毒素I的氨基酸组成。 试验结果还表明,用13％胶浓度的SDS-不连续聚丙烯酰胺凝胶板状电泳测定小于10,000道尔顿的蛋白质的分子量,可以获得较为满意的结果。     

用二次电泳法研究核酸与蛋白质的相互作用

研究蛋白质与核酸的结合常遇到的问题是对蛋白质等电点及可溶度等要求较高,或难以同时处理大量标本。为克服此缺点,将待检蛋白经聚丙烯酰胺凝胶电泳后,通过洗涤去除凝胶中的ＳＤＳ,使蛋白质相对固定于凝胶中,改电泳液为ＴＡＥ或ＴＢＥ,继之用同位素标记寡核苷酸进行二次电泳,通过放射自显影直观地显现出蛋白结合核酸的结果。该法敏感,特异,对蛋白质等电点及可溶性要求低,可同时检测多个样本,值得推广使用。     

不经染色直接从SDS-PAGE制备凝胶中准确切下所需蛋白条带的方法

蛋白样品的垂直板SDS-聚丙烯酰胺凝胶电泳 (SDS-PAGE)不但是一种最常用的蛋白分析方法,也经常用于蛋白质的制备。从电泳凝胶上纯化蛋白,一般都要先用考马斯亮蓝染色,然后切下所需的蛋白条带。这里介绍一种可以不染色,直接从SDS-PAGE制备凝胶上准确切割所需蛋白条带的方法。与染色后切胶的方法相比,这种方法简单、省时,分离到的蛋白容易从胶中洗脱回收,并可明显提高回收率,而且省去了令人烦怖的从回收的蛋白中脱去染色时结合的染料的问题。作者曾用此方法分离过多种蛋白,屡试不爽。这种方法与一般的SDS-PAGE制备电泳的差别主要在电泳结束后对凝胶的处理上。     

一种改进的双向电泳染色方法

本文涉及了双向电泳过程中的染色方法,即先用考马斯亮蓝染色,将胶上可见蛋白切下再银染的方法。这种方法可最大限度的减少胶中蛋白质点的损失,不仅避免了单一用考马斯亮蓝染色由于灵敏度不高而导致的低丰度蛋白的损失,也避免了单一用银染而使高丰度的蛋白因染色过度导致的损失。同时两种传统的染色方法结合完美,形成的新方法经济实用。     

二维电泳分离牛精子蛋白的技术研究

二维电泳是蛋白质分离技术并可由于对精子蛋白的分离。本研究旨在通过对双向电泳条件的研究摸索出一种适用于分离牛精子蛋白的二维电泳技术,并利用其对牛精子蛋白进行分离鉴定。在实验中,优化了等电聚焦程序,研究了精子蛋白的不同制备方法、不同上样量、不同胶条长度对电泳结果的影响。结果表明,采用尿素－盐酸胍两步裂解法裂解精子细胞制备蛋白,使用13cm非线性胶条进行蛋白二维电泳,能获得较好的电泳图谱。图谱经二维电泳软件分析,可检测出约800多个蛋白质点,分子量基本分布在10~100KD、等电点约为4~9的区域内。对精子蛋白二维电泳条件的摸索,为后续牛精子X、Y差异蛋白的检测和分析奠定了理论基础。     

Comparative analysis of protein aggregates by blue native electrophoresis and subsequent sodium dodecyl sulfate-polyacrylamide gel electrophoresis in a three-dimensional geometry gel

We describe the comparative analysis of protein aggregates by combining blue native electrophoresis and subsequent sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using a 3-D geometry gel for simultaneous processing of many samples. The first native electrophoresis step, separating the aggregates, is carried out for a series of samples in parallel lanes within a slab gel. This gel is then placed on the top surface of a cylindrical, 3-D geometry gel for the second denaturing electrophoresis step, separating the proteins composing the aggregates. The samples migrate parallel to the vertical axis of the gel cylinder. Data are acquired online by photodetection of laser-induced fluorescence during electrophoresis. For this purpose, the samples are fluorescently labeled within the slab gel after the first separation step. A 3-D geometry gel separates the equivalent of many conventional SDS slab gels represented by vertical layers in the 3-D gel body. In this way, many samples are analyzed in the same gel under identical conditions, improving comparability and resolution and making the process considerably more efficient. This novel technique allowed the identification of several aggregate classes of recombinant proteins expressed in bacteria. We observed that proteins preferentially bind to homolog polypeptides, but also seem to form a trapping mesh co-aggregating with other proteins. The aggregation pattern revealed by this technique supplements data obtained from standard two-dimensional gel electrophoresis analysis. We expect interesting applications, for instance in aggregate monitoring of clinical samples. It should be feasible to quickly gain a diagnostic picture during amyloid-related neurodegenerative disease development or to observe drug effects on protein aggregation.     

Analysis of mitogenic activity of proteins after separation by gel electrophoresis

We have used a combination of gel electrophoresis and a cell culture assay in microplates to analyse mitogenic activity in tissue extracts. The procedure is a modification of the method described by Kuo et al. The proteins were separated by native gel electrophoresis or isoelectric focusing. The gel was sliced and defined pieces were transferred into tissue culture inserts fitting in 96 well microplates, which contained the test cells. The proteins diffused from the gel slices directly into the culture supernatant and the mitogenic effects were evaluated by a colorimetric assay (MTT or phosphatase activity). Human interleukin 2 was used to demonstrate the feasibility of the method by evaluating the mitogenic effect on the cell line CTLL-2. Extracts of bovine pituitary glands were separated by native gel electrophoresis and isoelectric focusing and several protein bands could be identified which showed a distinct mitogenic effect on human endothelial cells. The method is very sensitive and allows rapid screening of protein mixtures for bioactive fractions. This revised version was published online in July 2006 with corrections to the Cover Date.     

小麦幼穗蛋白质双向电泳条件的优化

本研究以温光敏小麦为试材,用TCA/丙酮和酚提取法提取小麦幼穗蛋白样品,进行了双向电泳优化分析,并对双向电泳过程中出现的问题进行了讨论。结果表明,用TCA/丙酮法提取小麦幼穗蛋白质其产率(浓度)高于酚提取法。SDS-PAGE电泳显示,用TCA/丙酮提取法提取的蛋白质能获得较清晰条带,分辨率较高,而酚提取法提取的蛋白质其条带模糊,分辨率低。对蛋白质纯化除盐可以提高分辨率,减少横竖纹,获得背景清晰的圆形蛋白点。通过ImageMasterTM 2D Platinum5.0软件分析凝胶图谱,结果显示纯化后可降低噪点,纯化后蛋白点数可从未纯化蛋白点数的216增加到583。显然,采用TCA/丙酮法可获得高浓度高质量的蛋白质,而进一步纯化、除盐离子可进一步获得背景清晰可高重复性的电泳图谱。在双向电泳实验过程中,观察到一些异常缺陷胶的出现,如双向电泳图谱中蛋白点扩散,蛋白聚集形成斑点串,没有点或点很少,出现纵纹横纹及图谱扭曲等影响图谱质量的严重问题,本研究对这些问题做了分析并提出了解决方案。     

Pouring and Running a Protein Gel by reusing Commercial Cassettes

The evaluation of proteins using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis is a common technique used by biochemistry and molecular biology researchers^1-4. For laboratories that perform daily analyses of proteins, the cost of commercially available polyacrylamide gels (˜$10/gel) can be considerable over time. To mitigate this cost, some researchers prepare their own polyacrylamide gels. Traditional methods of pouring these gels typically utilize specialized equipment and glass gel plates that can be expensive and preclude pouring many gels and storing them for future use. Furthermore, handling of glass plates during cleaning or gel pouring can result in accidental breakage creating a safety hazard, which may preclude their use in undergraduate laboratory classes. Our protocol demonstrates how to pour multiple protein gels simultaneously by recycling Invitrogen Nupage Novex minigel cassettes, and inexpensive materials purchased at a home improvement store. This economical and streamlined method includes a way to store the gels at 4°C for a few weeks. By re-using the plastic gel cassettes from commercially available gels, labs that run frequent protein gels can save significant costs and help the environment. In addition, plastic gel cassettes are extremely resistant to breakage, which makes them ideal for undergraduate laboratory classrooms.     

Molecular characterization of proteinaceous material in the Florida coastal Everglades

Dissolved organic nitrogen (DON) is the least known component of the nitrogen cycle, in part as a result of the lack of adequate analytical methods for its molecular characterization. In this study proteinaceous material in DON, collected at six geomorphologically different sites in the Florida coastal Everglades, was characterized by amino acid analysis and protein gel electrophoresis. The amino acid composition of the samples suggests that the canal DON was more degraded and subject to higher microbial inputs than the mangrove marshwater and marine end-member stations. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) results supported this observation as distinctly different protein profiles were obtained for the canal waters compared to samples collected at other stations. These preliminary results highlight the potential of combining amino acid and intact protein analysis to fingerprint the sources of DON in different aquatic environments, and show SDS-PAGE as a potentially useful method to characterize DON.     

Molecular weights of subunits of acetyl CoA carboxylase in rat liver cytoplasm

Monomeric [14C] methyl avidin was shown to bind to sodium dodecyl sulfate-denatured biotinyl proteins and remain bound through polyacrylamide gel electrophoresis which allowed their detection by fluorography. This method was used to show that purified rat liver acetyl CoA carboxylase contained two high molecular weight forms of the enzyme (MR = 241,000 and 252,000) while rapidly prepared, crude rat liver cytoplasm contained two larger molecular weight (MR = 257,000 and 270,000) forms. Thus, the enzyme had undergone substantial proteolysis during purification. The crude enzyme preparation also contained a smaller biotinyl protein (MR = 141,000) which is likely a proteolytic product of the larger forms of acetyl CoA carboxylase.     

Biochemical characterization of six trisomics of grain sorghum,Sorghum bicolor (L.) Moench

To determine protein differences of grain sorghum disomics and trisomics, we analyzed leaf extracts from six trisomics and a disomic control by disc gel, gel isoelectric focusing, and SDS gel electrophoresis. Based on the number and position of protein bands revealed by Commassie blue staining, the disomic control could be differentiated from the trisomics, and trisomics could be shown to differ among themselves in most cases. SDS gels revealed the most protein bands, followed by isoelectric focusing and disc gel. However, disc gel electrophoresis was the simplest technique of the three and was just as effective in identifying trisomics and differentiating trisomics from the disomic control.Contribution 1596-j, Department of Agronomy, and 182-j, Department of Biochemistry, Kansas State University, Manhattan, Kansas.     

茶树叶片蛋白双向电泳体系建立与应用

为开展茶树Camellia sinensis 低温和干旱胁迫下差异蛋白的分离和鉴定,以抗逆性较强的茶树品种‘迎霜’为试材,通过对提取方法、IPG 胶条pH 范围、上样量、分离胶浓度、染色方法的比较,筛选适用于茶树叶片的蛋白质双向电泳体系。结果表明,采用TCA-丙酮法或Tris-HCl 法提取叶片总蛋白,选用17 cm pH 4~7IPG 胶条用于等电聚焦,选择1.6~2.2 mg 上样量、13.5%聚丙烯酰胺凝胶进行分离,随后通过高敏考马斯亮蓝R-250 法染色;最终,叶片各分子量的蛋白充分分离,获得的双向电泳图谱分辨率高、背景清晰、重复性好,适用于‘迎霜’低温和干旱胁迫下叶片差异蛋白分析。