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.     

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。     

Immunolocalization and Western Blot Analysis of Nitrogenase in Oscillatoria limosa During a Light-dark Cycle

Nitrogenase of the non-heterocystous nitrogen-fixing cyanobacterium Oscillatoria limosa was subjected to western blot analysis and immunogold electron microscopy using antisera raised against dinitrogenase (MoFe-protein, Component I) and dinitrogenase reductase (Fe-protein, Component II). O. limosa was grown diazotrophically under an alternating light-dark cycle (16–8h light-dark). Although nitrogenase activity (acetylene reduction) was found predominantly during the dark phase, being absent during most of the light period, immunogold electron microscopy revealed label of both subunits of nitrogenase in samples taken throughout the light-dark cycle. It was also shown that the nitrogenase label was distributed homogeneously in the cell and that it was present in every cell of every trichome whether fixing nitrogen or not. On average, 34 (± 6) gold particles μm^?2 thin section were detected. Nitrate-grown cells did not contain nitrogenase label. Western blot analysis of the Fe-protein in samples taken during the light phase, revealed a single band with an apparent molecular weight of 37 kDa. At the end of the light period, and during the dark phase when high nitrogenase activities were observed, an additional band of 36 kDa was found. The anti-MoFe-protein antiserum revealed a single band of 56 kDa which was present throughout the light-dark cycle. Nitrate-grown cells were not recognized by either antiserum. It is concluded that nitrogenase enzyme is present in O. limosa throughout the light-dark cycle but that the Fe-protein is modified (inactive form) during the light period when nitrogenase activity is absent.     

用蛋白质印迹转移技术分析正常及硒性白内障大鼠晶状体及房水中蛋白质的性质

本文用蛋白质印迹转移技术分析了正常及硒性白内障大鼠晶状体及房水中蛋白质的性质。结果表明,晶状体中的脲溶性蛋白质可被抗α及抗γ晶体蛋白血清识别,提示α及γ晶体蛋白均为脲溶性蛋白质的主要成份。患白内障时房水中的蛋白质含量明显增加,且主要被抗γ血清识别,而被抗α血清识别的成份很少,表明在大鼠硒性白内障形成过程中,有较多低分子量蛋白质漏出到房水中,且其主要成份为γ晶体蛋白。此外,我们还发现正常及硒性白内障大鼠晶状体膜蛋白质与抗α及抗γ血清起反应的程度及分布有所不同,提示晶状体细胞膜与晶体蛋白之间存在着相互作用。     

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。     

蛋白免疫印迹法同时检测大、小分子蛋白的实验条件改进

目的:蛋白免疫印迹法是现代生物医学研究中广泛应用于蛋白定性和半定量分析的实验技术。然而,常规采用传统单一浓度凝胶的蛋白免疫印迹法在应用过程中仍有不足之处,如不能同时检测分子量很大和很小的蛋白,因而有必要探索一种增大凝胶有效分离范围的检测方法。本文提出采用组合凝胶来实现更大范围分子量蛋白的同时检测。方法:比较双浓度的组合凝胶与单一浓度凝胶的分离范围以及分析采用组合凝胶,蛋白免疫印迹法对大、小分子蛋白的检测效果。结果:12%/7.5%组合凝胶和15%/7.5%组合凝胶的分离范围显著大于相应的单一浓度凝胶。通过12%/7.5%组合凝胶,蛋白免疫印迹法同时检测到15-300 k Da范围内的大、小分子蛋白。结论:组合凝胶有助于蛋白免疫印迹法对分子量相差很大的蛋白进行同时检测分析,具有很强的实用性。     

Leaf Abscission in Soybean: Cytochemical and Ultrastructural Changes following Benzylaminopurine Treatment

6-benzylaminopurine (BAP) delays leaf abscission of soybeanGlycine max (L.) Merr. Abscission of the distal pulvinus ofprimary leaves was induced in 12-d-old seedlings or explantsby removal of the leaf blade. BAP applied to the cut end ofthe pulvinus following leaf blade removal delayed abscission.Discoloration of the pulvinus occurred before abscission commencedand the number of grana in chloroplasts within cortical parenchymacells of the pulvinus decreased over time following leaf bladeremoval. BAP prevented discoloration of pulvinus tissues anda decrease in grana number. Starch grains within amyloplastsof cells of the starch sheath in the pulvinus disappeared followingleaf blade removal, whereas starch accumulated within the abscissionzone prior to abscission. BAP prevented this apparent redistributionof starch and instead promoted an increase in starch withinplastids of cortical parenchyma cells of the pulvinus. Duringthe abscission process, cells within the separation layer enlargedand their nuclei and nucleoli became more evident prior to theirseparation from one another. Cell separation resulted from breakdownof middle lamellae and partial degradation of primary cell walls.Cycloheximide applied directly to the external surface of theabscission zone inhibited abscission in a similar way to theBAP treatment. These results suggest that BAP prevents abscissionby altering patterns of starch distribution in the pulvinusand abscission zone and by inhibiting the synthesis of proteinsthat typically appear de novo in induced abscission zone tissues. Key words: Benzylaminopurine, BAP, Soybean, Pulvinus, Abscission, amyloplast.     

一种转移非特异性蛋白带的染色方法

考马斯亮蓝是常用的聚丙烯酰胺凝胶蛋白电泳的染料,利用硝酸纤维素膜(NCF)对染料的吸附作用,将低浓度的考马斯亮蓝(0.025%)染色液直接对NCF上的转移蛋白带进行染色,经实验反复验证.它是一种较好的NCF上转移非特异性蛋白带的染色方法.     

单纯疱疹病毒糖蛋白D的表达及免疫学鉴定

单纯疱疹病毒(herpes simplex virus,HSV)是TORCH综合征的病原体之一.新生儿可通过宫内、产道和出生后等多种途径感染,大部分患儿呈现症状,如皮炎、角膜炎、口唇疱疹,也可发生涉及多个器官的播散性感染,严重者出现疱疹性脑膜炎,并常导致婴幼儿死亡.目前尚无全身用的特效药物和有效的防范措施.HSV包膜糖蛋白D(glycoprotein D,gD)是极为保守的免疫原性蛋白,在体内可诱导高滴度的中和抗体,因此gD基因成为近些年来诊断研究的靶基因.本文尝试将gD蛋白在酵母菌中表达,并分析其抗原性,为建立快速易行的重组抗原诊断试剂盒奠定基础.此外,利用该表达系统表达的HSV gD蛋白,可为HSV基因工程重组疫苗的研制提供依据,对优生优育、提高人口出生质量具有重要的理论及实际意义.     

大豆初生幼苗多胺氧化酶活性的细胞化学定位

对大豆(Glycinemax(L.)Merril“l)垦农4号”萌发种子和初生幼苗中的多胺氧化酶(polyamineoxidase,PAO,EC1.4.3.6)的活性和分布进行了研究。结果表明,PAO活性仅在种子萌发起始后(吸胀后24h)才检测到,然后随着种子萌发进程,PAO活性快速升高。但是,在萌发种子(吸胀后72h)和初生幼苗(吸胀后120h)中,PAO活性在各器官中的分布有明显差异。在萌发种子中,PAO活性在胚根最高(5.17±0.91Ug-1FW),胚轴次之,胚芽再次之,子叶活性最低(0.12±0.03Ug-1FW);在初生幼苗中,PAO活性在下胚轴中最高(5.47±0.66Ug-1FW),幼根次之,顶芽再次之,子叶最低(0.10±0.03Ug-1FW)。这种差异对种子萌发和幼苗形态建成有积极意义。运用细胞化学定位在透射电镜下观察初生幼苗PAO在各部位的分布,发现PAO主要定位在顶芽细胞的液泡膜上、子叶细胞的细胞壁及其外侧表面、下胚轴细胞的细胞壁及其表面,且PAO与细胞壁表面结合较紧;根细胞的细胞壁、细胞间隙、细胞膜、液膜上均有分布,但以液泡膜分布居多。本研究结果进一步证实了PAO在细胞壁和细胞间隙有着较广泛的分布。首次报道PAO在细胞膜和液泡膜上有分布。     

Evaluating Strategies to Normalise Biological Replicates of Western Blot Data

Western blot data are widely used in quantitative applications such as statistical testing and mathematical modelling. To ensure accurate quantitation and comparability between experiments, Western blot replicates must be normalised, but it is unclear how the available methods affect statistical properties of the data. Here we evaluate three commonly used normalisation strategies: (i) by fixed normalisation point or control; (ii) by sum of all data points in a replicate; and (iii) by optimal alignment of the replicates. We consider how these different strategies affect the coefficient of variation (CV) and the results of hypothesis testing with the normalised data. Normalisation by fixed point tends to increase the mean CV of normalised data in a manner that naturally depends on the choice of the normalisation point. Thus, in the context of hypothesis testing, normalisation by fixed point reduces false positives and increases false negatives. Analysis of published experimental data shows that choosing normalisation points with low quantified intensities results in a high normalised data CV and should thus be avoided. Normalisation by sum or by optimal alignment redistributes the raw data uncertainty in a mean-dependent manner, reducing the CV of high intensity points and increasing the CV of low intensity points. This causes the effect of normalisations by sum or optimal alignment on hypothesis testing to depend on the mean of the data tested; for high intensity points, false positives are increased and false negatives are decreased, while for low intensity points, false positives are decreased and false negatives are increased. These results will aid users of Western blotting to choose a suitable normalisation strategy and also understand the implications of this normalisation for subsequent hypothesis testing.     

Telomere Length Distribution and Southern Blot Analysis

Southern blot analysis of terminal restriction fragments (TRFs) is the standard method for quantitative examination of telomere length distributions. Since TRFs contain a subtelomeric component, central parameters of the TRF distributionn(L) such as the arithmetic mean (M) or the median (Me) cannot be derived directly from Southern blot data, i.e. from the optical density distributionOD(L). Several estimates have been applied instead; the seeming arithmetic meanA, the “center of mass”C, and the positions of maximal (P) and half-maximal optical density (P). We show thatC>A>Mfor any non-truncated distributionsn(L), andP>M>P1/2 for any symmetrical unimodaln(L).Symmetric appearance on a Southern blot, however, suggests positive skewness ofn(L). Thus, alognormalform ofn(L) may be considered. Then,C>A>M>P=Me>P. Alternatively, aWeibulldistribution may be assumed. The latter is compatible with negative feedback-regulation of the telomere lengths. Using the maximum likelihood method we compare these distributions with FISH-data on telomere lengths in different cell types. The fit of the lognormal distribution is clearly superior. Lognormal genesis may relate to telomere breakage and recombination.Truncation of the upper end of the TRF distribution is possible due to Southern blot artifacts. Thereby, the order of the estimates may change toP>C>A. Having minimal sensitivity to truncation,Pseems to be the optimal choice. however, the variability ofPis high since peakedness ofOD(L) and DNA length resolution are inversely related.     

HIV-1抗体蛋白印迹确认与核酸检测复核对比研究

应用病毒核酸载量法NASBA和HIV-1 RNA的巢式逆转录PCR(nested RT-PCR)法与HIV抗体蛋白印迹(WB)方法,对经过初筛的44例HIV-1抗体阳性标本进行了对照检测研究。发现了2例(gp160、p24)和1例(gp160g、p120p、66、p24)的特殊阳性样本,经NASBA法和该RT-PCR法核酸检测为阴性;WB确认的4例gp160阳性带、1例p24、p17阳性带和13例p24阳性带,经NASBA法和该RT-PCR法核酸检测也为阴性;而WB确认的其余全部带型的抗体阳性标本经过NASBA法和该RT-PCR法检测均为阳性。该研究表明对只有gp160p、24和gp160、gp120p、66、p24的特殊阳性标本和以p24为主的抗体不确定标本需要用RT-PCR或NASBA方法进行核酸检测,以进一步确认。     

Analysis of Abscisic Acid in the Brains of Rodents and Ruminants

The surface tension (σ) of batter prepared with one or two of the major ingredients of wheat flour, egg and sucrose was measured by the maximum bubble pressure method. The surface tension of the soft- and hard-wheat flour suspension decreased as the solid content of wheat flour was increased, finally reaching 48.2 and 52.7 dyn/cm, respectively. The surface tension of the wheat flour suspension was lower than that of its supernatant, which suggests that the precipitate reduced the surface tension. Gelatinization of the wheat flour suspension lowered its surface tension. The surface tension of the whole egg dispersion decreased markedly to about 53 dyn/cm as the solid concentration was increased from 0 to 1%, and remained almost constant as the concentration was increased further. This tendency was almost the same as that of the egg yolk dispersion. The surface tension of mixtures of two ingredients such as egg and wheat flour, and egg and sucrose was almost equal to that of the ingredient with the lower surface tension at the same concentration as in the mixture.     

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.