Western immunoblotting: temperature-dependent reduction in background staining

The effect of incubation temperature on the background staining of Western blots with monoclonal antibodies to a human milk protein, alpha-lactalbumin (Mr 14,500), is presented. Human milk proteins were electrophoretically separated and transferred to nitrocellulose membranes which were then blocked with bovine serum albumin, "BLOTTO", casein, or Tween 20. They were subsequently incubated with mouse monoclonal antibody to human alpha-lactalbumin, biotinylated anti-mouse antibody, strepavidin-biotinylated horseradish peroxidase complexes and a substrate containing diaminobenzidine and nickel chloride. Reduction of incubation temperature from 37 degrees C to 22 degrees C and 4 degrees C was found to decrease the extent of non-specific background staining independent of the type of blocking reagent used. Good specific staining with minimal background was found using 0.1% Tween 20 in phosphate-buffered saline, pH 7.2, as blocking agent and incubation temperatures of 4 degrees C.     

Western Blotting Inaccuracies with Unverified Antibodies: Need for a Western Blotting Minimal Reporting Standard (WBMRS)

Western blotting is a commonly used technique in biological research. A major problem with Western blotting is not the method itself, but the use of poor quality antibodies as well as the use of different experimental conditions that affect the linearity and sensitivity of the Western blot. Investigation of some conditions that are commonly used and often modified in Western blotting, as well as some commercial antibodies, showed that published articles often fail to report critical parameters needed to reproduce the results. These parameters include the amount of protein loaded, the blocking solution and conditions used, the amount of primary and secondary antibodies used, the antibody incubation solutions, the detection method and the quantification method utilized. In the present study, comparison of ubiquitinated proteins in rat heart and liver samples showed different results depending on the antibody utilized. Validation of five commercial ubiquitin antibodies using purified ubiquitinated proteins, ubiquitin chains and free ubiquitin showed that these antibodies differ in their ability to detect free ubiquitin or ubiquitinated proteins. Investigating proteins modified with interferon-stimulated gene 15 (ISG15) in young and old rat hearts using six commercially available antibodies showed that most antibodies gave different semi-quantitative results, suggesting large variability among antibodies. Evidence showing the importance of the Western blot buffer and the concentration of antibody used is presented. Hence there is a critical need for comprehensive reporting of experimental conditions to improve the accuracy and reproducibility of Western blot analysis. A Western blotting minimal reporting standard (WBMRS) is suggested to improve the reproducibility of Western blot analysis.     

Minimizing ELISA background in the diagnosis of swine trichinellosis.

After confirming that long-term serum storage (frozen at -20 C for greater than 3 mo) causes optical density to drift upward, several modifications of an enzyme-linked immunosorbent assay (ELISA) protocol were evaluated to identify a protocol that would reduce background in porcine sera tested for trichinellosis. Modifications evaluated included blocking the antigen-coated ELISA plate with sample diluent containing 10% bovine serum albumin (BSA) or 10% nonfat milk powder (bovine lacto transfer optimizer or BLOTTO), diluting sera in sample diluent containing 10% BSA or 10% BLOTTO, and preincubating samples in sample diluent containing 10% BSA or 10% BLOTTO. Overnight preincubation (approximately 12 hr at 2 C) of fresh sera diluted (1:10) in sample diluent containing 10% BLOTTO significantly reduced background and improved the detection of experimentally infected pigs by enhancing positive-negative discrimination. When testing stored sera, the modified protocol effectively reduced the effect of storage and the kit revealed specificity of 98.4%; there was no loss in sensitivity. The effect of long-term storage at -20 C must therefore be considered when testing swine sera for trichinellosis by ELISA and possibly also when conducting other immunoglobulin assays. The modification described here may prove useful if there is no alternative to using serum stored for greater than 3 mo at -20 C.     

Rapid high resolution western blotting: from gel to image in a single day.

A streamlined protocol is described that allows high sensitivity antigen detection by Western blotting in a single day. The choice of membrane blotting matrix, as well as blocking reagents, has been optimized in order to allow rapid development of the blot with chemiluminescent reagents. The entire process, from gel to blot to a permanent, hard copy image on x-ray film, can be accomplished within six hours.     

Genetic analyses of restriction fragment length polymorphisms using high molecular weight DNA from sperm or lymphocytes embedded in agarose

A simple and efficient method for determining restriction fragment length polymorphism types on large numbers of individuals using small samples of peripheral blood or sperm cells is described. Whole cells embedded in low gelling/melting temperature agarose were treated with a series of enzyme, detergent, and washing steps to release high molecular weight DNA that was then digested with standard restriction enzymes such as EcoRI and PstI, electrophoresed, blotted, and probed as in normal Southern analyses. The technique should be readily adaptable to any application requiring DNA from small numbers of cells for Southern analyses or pulsed field gel electrophoresis.     

Optimization of immunogold labeling TEM: an ELISA-based method for evaluation of blocking agents for quantitative detection of antigen.

We developed an ELISA-based method for rapid selection of optimal blocking agents to be used in antigen quantification by immunogold labeling electron microscopy. Casein, skim milk, BSA from two sources, acetylated BSA, fish skin gelatin, horse serum, and goat serum were tested for their ability to block nonspecific binding of antibody to recombinant Vitreoscilla hemoglobin (VHb) antigen expressed in Escherichia coli cells by ELISA and the results were confirmed by quantitative immunogold labeling transmission electron microscopy (TEM). Ability to minimize NSB was also evaluated by dot-blot and Western blotting methods. The results demonstrated that ELISA was most accurate in predicting the most efficient blocking agent for TEM. Existing methods could not provide an accurate picture of the ability of various reagents to suppress background labeling. The sensitivity of detection of antigens by immunoelectron microscopy depends on the assay procedure being optimized to obtain the highest possible signal along with as low a background (noise) as possible. Our study indicated that an ELISA-based evaluation of various blocking agents could help in the rapid selection and optimization of a suitable protocol for immunogold localization and quantification of antigens by TEM.     

Glutaraldehyde fixation increases retention of low molecular weight proteins (growth factors) transferred to nylon membranes for western blot analysis

Western blotting of low molecular weight (Mr) acidic and basic isoelectric point (pI) proteins was studied to optimize detection sensitivities. Radioiodinated epidermal growth factor (EGF, Mr 6045, pI 4.4), transforming growth factor type alpha (TGF alpha, Mr 5623, pI 6.8), insulin-like growth factor (IGF-I, Mr 7649, pI 8.3), and basic fibroblast growth factor (bFGF, Mr 15,000-17,000, pI 9.6) all transferred with high efficiency (74.1 +/- 12.6%) to a positively charged nylon membrane. Sequential application of standard unoccupied site blocking, antibody incubation, and washing steps resulted in significant losses of all growth factors (46-98%). Basic FGF was retained best. Treatment of transfer membranes with 0.5% (v/v) glutaraldehyde prior to blocking and immunodetection increased the retention of the growth factors 1.5- to 12-fold over untreated controls. Without fixation, 100 ng of EGF, TGF alpha, and IGF-I were not detectable while 6.25-100 ng was identified on fixed membranes. The methods described were equally sensitive for detecting both acidic and basic pI proteins.     

Analysis of complex autoantibody repertoires by surface-enhanced laser desorption/ionization-time of flight mass spectrometry

Normal sera contain a large number of naturally occurring autoantibodies which can mask important disease-associated ones. Western blotting has evolved as the most important tool to demonstrate autoantibodies in autoimmune diseases, because of its ability to simultaneous screening for a wide spectrum of different antigens. In previous studies we have shown the diagnostic potential of the analysis of autoantibodies in autoimmune diseases by means of multivariate statistics and artificial neural networks. However, the Western blotting procedure remains very time-consuming and is also limited in sensitivity. Therefore, we used an on-chip approach for the analysis of autoantibodies. This ProteinChip system uses ProteinChip arrays and SELDI-TOF MS (surface-enhanced laser desorption/ionization-time of flight mass spectrometry) technology for capturing, detection, and analysis of proteins without labelling or without the need of chemical modification. The microscale design of the arrays allows the analysis of very small quantities of proteins. In the present study, we used arrays with biologically activated surfaces that permit antibody capture studies. Protein-A-Chips were incubated with sera of patients (n = 12). After washing, the chips were incubated with a complex solution of autoantigens and subsequently washed again. If the Protein-A bound autoantibodies recognized their antigens, these proteins could be separated by their molecular masses and were to be detected by mass spectrometry. Previous studies using monoclonal antibodies have demonstrated that the detection limit is in the attomole level. Furthermore, all sera were analyzed by conventional Western blotting for direct comparison. In the present study, we have shown complex on-chip antibody-antigen reactions. At higher molecular weights (> 30 kDa) the detection sensitivity of this on-chip method was comparable to conventional Western blotting. At lower molecular mass, the Western blot technique is easily exceeded by the on-chip method. Considering that this on-chip procedure is quite easy to use, is much less time-consuming than Western blotting, and is much more sensitive at least in the low molecular weight range, the SELDI-TOF technology is a very promising approach for the screening of autoantibodies in autoimmune diseases. Due to its versatility, this on-chip technology could allow the large-scale screening for complex autoantibody distributions for diagnostic purposes and early detection of autoimmune diseases might be possible.     

Identification of two major families of transferrin receptors among Neisseria meningitidis strains based on antigenic and genomic features

Abstract The transferrin receptor or transferrin-binding proteins (Tbps) of 50 strains of Neisseria meningitidis belonging to different serogroups were examined by Western blotting using two rabbit antisera raised against Tbp purified from N. meningitidis strains B16B6 and M982. On the basis of the reactivity of Tbp2 with the antisera two patterns were observed and allowed the classification of 74% of the strains in group I (M982-like strains) and 26% in group II (B16B6-like strains). Southern blot analysis was performed on the genomic DNA of 16 meningococcal strains and showed that under stringent conditions, the tbp2 probes were specific for each group identified. Both immunological and genomic analyses have led to the identification within N. meningitidis strains of two major families distinguished on the basis of the characteristics of Tbp2 molecules, independently of serogroup, type or subtype.     

Application of SYPRO Ruby- and Flamingo-stained polyacrylamide gels to Western blot analysis

Western blot analysis has been a useful method for analysis of expression levels of specific proteins and is conducted after sodium dodecyl sulfate (SDS) or native polyacrylamide gel electrophoresis without staining the gel. However, when it is necessary to analyze the gel, duplicate polyacrylamide gels usually must be prepared, one of which is stained, leading to the consumption of precious sample. Thus, we developed a convenient and efficient Western blotting method using a stained gel. This simple modification should be beneficial for analyzing samples that are limited in quantity and/or samples for which the stained gel serves as the loading control.     

Identification and characterization of the tumor suppressor p53 in channel catfish (Ictalurus punctatus)

Herein is presented the sequence of a catfish full-length p53 cDNA obtained from a cloned B cell line cDNA library. Southern blot analyses determined that a restriction fragment linked polymorphism (RFLP) existed with PstI among outbred catfish. Western blot analyses demonstrated that, when compared to PBLs, the catfish leukocyte lines express higher levels of p53 protein. Additionally, the results of Western blot analyses and in vitro translation experiments suggest that the catfish leukocyte lines may produce truncated forms of p53 due to internal initiation.     

Enolase from the ectomycorrhizal fungus Tuber borchii Vittad.: biochemical characterization, molecular cloning, and localization

Enolase from Tuber borchii mycelium was purified to electrophoretical homogeneity using an anion-exchange and a gel permeation chromatography. Furthermore, the corresponding gene (eno-1) was cloned and characterized. The purified enzyme showed a higher affinity for 2-PGA (0.26 mM) with respect to PEP; the stability and activity of enolase were dependent of the divalent cation Mg2+. T. borchii eno-1 has an ORF of 1323 bp coding for a putative protein of 440 amino acids and Southern blotting analysis revealed that the gene is present as a single copy in T. borchii. The enzymatic activity and the mRNA expression level evaluated in mycelia grown either in different carbon sources, in pyruvate or during starvation were the same in all the conditions tested, while biochemical and Northern blotting analyses performed with mycelia at different days of growth showed T. borchii eno-1 regulation in response to the growth phase. Finally, Western blotting analysis demonstrated that enolase is localized only in the cytosolic fraction confirming its important role in glycolysis.     

Detecting multiple proteins by Western blotting using same-species primary antibodies, precomplexed serum, and hydrogen peroxide

Western blot detection of multiple proteins is challenged by the need to use antibodies from the same species and the harsh stripping methods that can remove protein or reduce protein antigenicity. Quenching using 27% hydrogen peroxide was developed as an alternative to stripping to inhibit horseradish peroxidase used to detect secondary antibodies. To detect two epitopes with same-species primary antibodies, quenching was followed by incubation in a precomplexed mixture of primary and secondary antibodies for the second epitope plus serum from that species. Both methods will be valuable in specific detection of multiple proteins by Western blotting, and will save time, valuable samples, and reagents.     

杜氏盐藻外源基因稳定表达系统的构建

建立了一种单细胞海水绿藻--杜氏盐藻(Dunaliella salina Teod.)的外源基因稳定表达系统.通过电激法将携带乙肝病毒表面抗原基因(HBsAg)和氯霉素乙酰转移酶基因(CAT)的质粒转入盐藻细胞内,CAT基因为筛选基因.PCR和Southern杂交结果显示,HBsAg基因已经整合到盐藻基因组中.Northern杂交结果表明,转化成功细胞内的该基因已转录成mRNA.HBsAgELISA和Western杂交检测证明,HBsAg蛋白在转化的盐藻细胞内稳定地表达.同时,PCR和Southern杂交显示,CAT基因也已整合到盐藻基因组中.且CATELISA检测证明,CAT蛋白在转化体中也已稳定地表达.进一步对转化盐藻进行无氯霉素筛选培养,60代后,HBsAg基因依然稳定地存在并表达.本实验第一次报道了外源基因在杜氏盐藻细胞内的稳定表达.     

An automated Western blot analysis using the phastsystem

We have developed a rapid, automated method for preparing Western blots of very small amounts of proteins, utilizing a commercially available electrophoresis system (Phastsystem, Pharmacia). This system has been adapted to transfer to nitrocellulose experimental samples that were initially separated in the same system by gradient-sodium dodecyl sulfate gel electrophoresis. The developing unit of the Phastsystem has permitted automation of all the necessary steps including incubation with antibodies, saturation of nonspecific binding sites, and washing. The total elapsed time for protein separation, transfer, and staining is about 6 to 7 h.     

An alternative nonradioactive method for labeling DNA using biotin

An alternative nonradioactive labeling method and a highly sensitive technique for detecting specific DNA sequences are described. The labeling method requires the "Klenow" fragment of DNA polymerase I and random hexanucleotides (synthesized or naturally extracted) as a primer for the production of highly sensitive DNA probes. The system has three main steps: (i) labeling of DNA with biotinylated 11-dUTP; (ii) detection of biotinylated DNA by a one-step procedure with streptavidin-alkaline phosphatase complex; (iii) blocking of background with Tween 20. Twenty attograms (2 X 10(-17) g) of pBR322 plasmid DNA was detected by dot-blot hybridization. Upon Southern blot hybridization, 7.4 fg (7.4 X 10(-15) g) of pBR322 HindIII DNA was detected using the biotinylated pBR322 plasmid DNA probe; 40.8 ag and 7.4 fg of lambda HindIII DNA were detected with the biotinylated whole lambda DNA probe by dot and Southern blot hybridization, respectively. Specific bands were also detected with the biotinylated argininosuccinolyase probe upon Northern blotting of mouse poly(A+) RNA. Further applications for in situ hybridization are also described.     

Spectra of molecular changes induced in DNA of Drosophila spermatozoa by 1-ethyl-1-nitrosourea and X-rays

Mutations induced in Drosophila spermatozoa at the alcohol dehydrogenase Adh locus by 1-ethyl-1-nitrosourea (ENU) were compared to X-ray-induced mutations using genetic tests for complementation, southern blotting, western blotting and northern blotting. 8 of 10 ENU-induced mutations complemented all known adjacent loci and were presumed to be intragenic. In contrast, 8 of 30 X-ray-induced mutations were intragenic. Southern blot analysis showed that 2 of 7 intragenic mutations induced by X-rays were altered at the Adh locus, whereas all 8 intragenic ENU mutants appeared normal. Western blot analysis showed 4 of 7 intragenic mutants induced by X-rays produced a detectable polypeptide; 1 of the 4 had normal molecular weight and charge. In contrast, 7 of the 8 intragenic mutants induced by ENU produced a polypeptide of normal molecular weight and charge. One ENU and two X-ray-induced mutants, which had normal southern blots and no detectable polypeptide, produced normal molecular weight mRNA by northern blots. The interpretation of these results is that in spermatozoa X-rays induce primarily deletions that either produce deficiencies of the Adh locus or nonsense mutations within the locus, whereas ENU induces primarily missense mutations. This forward mutation assay based on loss of enzymatic activity efficiently recovered a broad spectrum of mutations ranging from missense to intragenic deletions and multi-locus deficiencies. Only 3 of these 40 mutations produced a polypeptide detectable as an electrophoretic variant.