A Defined Methodology for Reliable Quantification of Western Blot Data

Chemiluminescent western blotting has been in common practice for over three decades, but its use as a quantitative method for measuring the relative expression of the target proteins is still debatable. This is mainly due to the various steps, techniques, reagents, and detection methods that are used to obtain the associated data. In order to have confidence in densitometric data from western blots, researchers should be able to demonstrate statistically significant fold differences in protein expression. This entails a necessary evolution of the procedures, controls, and the analysis methods. We describe a methodology to obtain reliable quantitative data from chemiluminescent western blots using standardization procedures coupled with the updated reagents and detection methods.     

一种改良型的免疫印迹法

目的:蛋白免疫印迹法是现代生物实验过程中运用最为广泛的实验技术,常规的免疫印迹法在应用过程中存在很多弊端,如浪费抗体等,因此非常有必要探索出一种新型的免疫印迹法,本文旨在探索一种能够节约抗体的免疫印迹实验方法。方法:将8只SD大鼠随机分常规组和改进组两组,每组4只,活取视网膜组织,进行组织匀浆、蛋白定量,取不同蛋白总量的匀浆变性液进行β-Tubulin的免疫印迹实验,比较两组之间β-Tubulin的蛋白表达量之间是否存在显著性差异,实验需重复三次。结果:不同总量蛋白的免疫印迹显示两组之间β-Tubulin的表达量并无显著性差异。但是,相比常规方法,改进法使用的抗体量更少,条带更容易检测得到。结论:改进后的免疫印迹法能有效的节约抗体,操作方便,实用性强。     

成人腹泻轮状病毒蛋白的免疫印迹分析

我们从腹泻病人便样中纯化了病毒,其结构蛋白组分按分子量大小分别为VP1(136K),VP2(113K),VP3(92K),VP4(84K),VP5(64K),VP6(47K),VP7(41K)。所有这些蛋白皆具有抗原性。WP6是B组轮状病毒的共同抗原。B组轮状病毒的每一结构蛋白与A组轮状病毒都无交叉免疫反应。另外注意到二例不同病人对VP6和VP7刺激产生的抗体水平不同。     

The Fastest Western in Town: A Contemporary Twist on the Classic Western Blot Analysis

The Western blot techniques that were originally established in the late 1970s are still actively utilized today. However, this traditional method of Western blotting has several drawbacks that include low quality resolution, spurious bands, decreased sensitivity, and poor protein integrity. Recent advances have drastically improved numerous aspects of the standard Western blot protocol to produce higher qualitative and quantitative data. The Bis-Tris gel system, an alternative to the conventional Laemmli system, generates better protein separation and resolution, maintains protein integrity, and reduces electrophoresis to a 35 min run time. Moreover, the iBlot dry blotting system, dramatically improves the efficacy and speed of protein transfer to the membrane in 7 min, which is in contrast to the traditional protein transfer methods that are often more inefficient with lengthy transfer times. In combination with these highly innovative modifications, protein detection using infrared fluorescent imaging results in higher-quality, more accurate and consistent data compared to the standard Western blotting technique of chemiluminescence. This technology can simultaneously detect two different antigens on the same membrane by utilizing two-color near-infrared dyes that are visualized in different fluorescent channels. Furthermore, the linearity and broad dynamic range of fluorescent imaging allows for the precise quantification of both strong and weak protein bands. Thus, this protocol describes the key improvements to the classic Western blotting method, in which these advancements significantly increase the quality of data while greatly reducing the performance time of this experiment.     

Western Blot Detection of Human Anti-Chikungunya Virus Antibody with Recombinant Envelope 2 Protein

Chikungunya virus (CHIKV), a tropical pathogen, has re-emerged and has massive outbreaks abruptly all over the world. Containing many dominant epitopes, the envelope E2 protein of CHIKV has been explored for the vaccination or diagnosis. In the present study, the antigenicity of a recombinant expressed intrinsically disorder domain (IUD) of E2 was tested for the detection of the antibody against CHIKV through western blot method. The gene of the IUD of E2 was inserted into 2 different vectors and expressed as recombinant GST-E2 and recombinant MBP-E2 fusion protein, respectively. Two kinds of fusion proteins were tested with 30 CHIKV patient sera and 30 normal sera, respectively. Both proteins were detected by 25 patients sera (83.3%) and 1 normal serum (3.3%). This test showed a relatively high sensitivity and very high specificity of the recombinant E2 proteins to be used as diagnostic antigens against CHIKV infection.     

Reverse Line Blot Macroarray for Simultaneous Detection and Characterization of Four Biological Warfare Agents

The need for a rapid detection and characterization of biowarfare (BW) agents cannot be over emphasized. With diverse array of potential BW pathogen available presently, rapid identification of the pathogen is crucial, so that specific therapy and control measures can be initiated. We have developed a multiplex polymerase chain reaction based reverse line blot macroarray to simultaneously detect four pathogens of BW importance viz. Bacillus anthracis, Yersinia pestis, Brucella melitensis and Burkholderia pseudomallei. The multiplex PCR utilizes 14 pairs of primers targeting 18 specific markers. These markers include genes which are genus specific, species-specific chromosomal sequences and virulence markers of plasmid origin. The assay was evaluated on various human, environment and animal isolates. The assay w successful in simultaneous detection and characterization of isolates of the four pathogens on as a single platform with sensitivity ranging from 0.3 pg to 0.3 ng of genomic DNA. The assay was able to detect 5 × 10² cfu/ml for B. anthracis, 8 × 10² cfu/ml for Yersinia sp., 1.4 × 10² cfu/ml for B. melitensis and 4 × 10² cfu/ml for B. pseudomallei.     

兔骨组织总蛋白的提取和在免疫印迹法中的应用

探讨快速有效的骨组织总蛋白提取法和利用该方法进行免疫印迹研究.采用单纯研磨法、单纯锤击法和锤击研磨法分别对兔骨组织蛋白进行提取,通过聚丙烯酰胺凝胶电泳对蛋白进行分离,利用免疫印迹法检测骨组织中beta-actin的表达,比较3种骨组织蛋白提取法.发现3种蛋白提取法均可满足免疫印迹研究要求,但是锤击研磨法提取的蛋白含量比单纯锤击法高,并能更好的保留49 kDa以下的蛋白,而且该操作法比传统的单纯研磨法更为方便、快捷,还能节省液氛用量.锤击研磨法可作为提取骨组织蛋白的一种理想方法.     

基因芯片数据分析过程:从原始数据到生物学意义

基因芯片实验要得到可靠的生物学结论,必须基于优化的实验设计和科学的数据分析。讨论了与基因芯片数据分析方法相关的实验设计方面的几个问题,简述了差异表达分析、聚类分析及功能富集分析等分析方法及其进展,并介绍了部分软件及应用。     

Differentiation of Yam Virus Isolates Using Symptomatology, Western Blot Assay, and Monoclonal Antibodies

Monoclonal antibodies (mAbs) were prepared against a yam mosaic virus (YMV) isolate from the Côte d'Ivoire. Symptomatology, Western immunoblotting, and ELISA were used to discriminate 69 isolates of YMV originating from different Dioscorea species and from various yam producing areas. These isolates induced two types of symptoms, were of four different electrophoretic mobilities and formed two serogroups. These results suggest that at least six differetit groups of isolates exist, three of which infect the main cultivated species in various geographical areas while two others were from unusual samples in our collection. An isolate from 'Pilimpikou Yam' from Central Burkina Faso was serologically distinct. It is concluded that there is a significant variability among yam virus isolates which is unrelated to the origin of the isolate (geographic or host species). It is suggested that precautions should be taken in order to avoid international exchange of infected material.     

Cytochemical and Western Blot Analysis of the Subcompartmentalization of the Acrosome in Rodents using Soybean Lectin

In the present study, the formation and development of the acrosome during spermiogenesis in four different rodent species (rat, mouse, hamster and guinea pig) was compared by means of cytochemical and blotting techniques using a lectin from soybean (SBA). This lectin recognizes specifically the acrosome of the four species at all steps of formation. At the ultrastructural level, SBA-binding pattern was similar in the acrosome of the rat, mouse and hamster. SBA preferentially labelled the electron-lucent area of the acrosome in early spermatids (Golgi and cap phases) and the outer region of the acrosome in mature spermatids (acrosome and maturation phase). The lectin binding pattern was more complex in the guinea pig acrosome. Three different subdomains can be established in the early acrosome of the guinea pig. The lectin bound the three subdomains but mainly a thin fold which spreads over the nucleus during the cap phase. In the acrosome phase, SBA strongly reacted with the principal segment. In contrast, no reactivity was observed in most of this segment in maturation phase spermatids. In this phase, SBA bound preferentially a thin area covering the dorsal region of the apical segment. Lectin blots of detergent-extracted testes indicated that SBA only recognizes proteins of high molecular weight (>100kD) in the four species studied. The results obtained in the present study suggest that the development of acrosomal subdomains is very similar in the mouse, rat and hamster but shows a more complex pattern in the guinea pig.     

Western Blot analysis of the antigens of Toxoplasma gondii recognized by human IgM and IgG antibodies

Western Blot analysis revealed that both IgM and IgG antibodies present in the sera of humans infected with Toxoplasma gondii recognize three major antigens with apparent m.w. of 32,000, 22,000, and 6000, respectively. In addition, IgG antibodies recognized at least 17 other antigenic components. After subcellular fractionation, enrichment of the three major antigens recognized by IgM and IgG antibodies by the membrane fraction was observed. Solubilization of membrane-enriched preparations with a mixture of sodium dodecyl sulfate and sodium deoxycholate did not reveal any new antigenic structures that reacted with IgM or IgG antibodies. Treatment of Toxoplasma lysate preparations and various fractions obtained after differential centrifugation with NaIO4 diminished the reactivity of the antigens with both IgM and IgG antibodies. Lipase treatment had no effect on the number or nature of antigens recognized by IgM antibody. Treatment with pronase and trypsin eliminated the 32,000 and 22,000 m.w. antigenic components detected by IgM antibodies, whereas such treatment had no effect on the 6000 m.w. component. Periodic acid-Schiff staining of polyacrylamide gels of Toxoplasma sonicates revealed the presence of three components corresponding to m.w. of 62,000, 45,000, and 6000, respectively. At least 15 components, including the 6000 m.w. component, directly bound concanavalin A.