An improved method for Southern DNA and Northern RNA blotting using a Mupid-2 Mini-Gel electrophoresis unit

An improved method for Southern DNA and Northern RNA blotting using the Mupid-2 Mini-Gel System is described. We get sharp and clear bands in Southern and Northern blotting after only 30 min short gel electrophoresis instead of the several hours large gel electrophoresis of conventional methods. The high electrical voltage with a pulse-like current of the Mupid-2 Mini-Gel System also allows reduction of the amount of formaldehyde, a harmful reagent, from the gel running buffer in RNA blotting. This minor modification of DNA and RNA blotting technique enables us to perform the complete experimental procedure more quickly economically in less space, than conventional Southern and Northern blotting, as well as using an extremely small amount of formaldehyde in RNA blotting.     

电转移中蛋白质的透膜现象及其对蛋白质印迹结果的影响

探讨了电转移中蛋白质的透膜现象及其对蛋白质印迹结果的影响.采用抗凋亡抑制蛋白-Survivin的抗体,对细胞裂解液进行蛋白质印迹.与常规操作方法不同之处是：在电转移的凝胶“三明治”中,重叠放置两张硝酸纤维素膜.电转移后,对两张膜同时进行免疫印迹.在特定的转移条件下,两张膜的免疫印迹都出现了Survivin蛋白的特异印迹带,证实了电转移中存在着蛋白质的透膜现象.转移时间、电流强度和蛋白质的分子质量,都是影响蛋白质透膜的相关因素.电转移中蛋白质的透膜,可以产生“印迹复制失真”的效应,从而最终影响蛋白质印迹定性和定量的结果.所得实验结果和结论,揭示了蛋白质印迹技术方法学中一个需要加以充分关注的问题,对于科学掌握和应用该技术具有积极作用.     

Detection of IgA and IgM antibodies to HIV-1 in neonates by radioimmune western blotting.

OBJECTIVE--To detect infection with HIV-1 by IgA and IgM response at birth in children born to HIV-1 seropositive mothers. DESIGN--Western blotting and radioimmune western blotting on stored sera from infected and uninfected babies born to HIV-1 seropositive mothers. Sera were pretreated to remove IgG. SETTING--Parma and Bologna, Italy. SUBJECTS--12 infected and five uninfected babies born to HIV-1 seropositive mothers and three babies born to seronegative mothers. MAIN OUTCOME MEASURES--Effectiveness of western blotting and radioimmune western blotting in detecting antibodies to HIV-1 gene products. RESULTS--With conventional western blotting we found IgA class antibodies to HIV-1 proteins in serum from three out of 12 infected children; in two of these three the serum was collected at age 3 months (positive controls). Radioimmune western blotting detected both IgA and IgM antibodies in serum from all infected children tested, whereas all serum from uninfected children born to seropositive and seronegative mothers showed no such antibodies. CONCLUSION--Although the technique should be tested on more patients, radioimmune western blotting seems to be a valuable tool for serological diagnosis of congenital HIV-1 infection at birth in neonates born to seropositive mothers.     

Valid application of western blotting

Western blotting is a powerful and commonly used tool to identify and quantify a specific protein in a complex mixture. However, the systematic errors in the application of western blotting analysis are frequently to be found, which may compromise the interpretation of results. To make a valid application of western blotting, it is essential to begin with three independent biological replicates. Subsequently, a more reliable normalization method is in urgent need for western blotting analysis and using reference proteins is the currently preferred method of normalization. Additionally, identification of valid reference proteins is crucial for western blotting analysis and it should be examined carefully in relation to the cell or tissue types when using housekeeping proteins as internal standards.     

用western blotting方法检测乙醇固定细胞中细胞周期素的表达

用western-blotting法测定乙醇固定细胞中细胞周期素（cyclin)的表达情况,探索western-blotting和流式细胞仪对固定细胞在流式细胞术分选前或分选后对同批标本进行蛋白同步分析的可行性,采用对数生长期的Molt-4细胞,经两种常规固定方法固定后,用western-blotting方法检测cyclinA,B1、D和E的表达,另取乙醇固定经流式细胞仪分选的G1期和G2/M期细胞裂解后,用western-blotting方法检测不同时相细胞中cyclinB1的表达。结果显示从固定细胞中提取的蛋白经western-blotting检测可得到清晰且分子量正确的条带,两种不同方法固定的细胞中检测到的cyclins表达未见明显差异。乙醇固定细胞经分选后提取蛋白行western-blotting检测可见G2/M期细胞的cyclinB1有明显表达而G1期细胞不明显。细胞进行固定,洗涤,染色和分选等处理后不影响western-blotting对其cyclins表达的分析。说明用western-blotting和流式细胞仪对固定细胞在流式细胞术分选前或分选后进行蛋白同步分析是可行的。     

A centrifugation method for determining the fresh weight of aquatic macrophytes

A centrifugation method to remove the surface water from five species of aquatic macrophytes of various morphologies is described and compared with the standard blotting method. The centrifugation method is up to twice as fast and up to three times more accurate than the blotting method. It works particularly well will plants of complex morphology and is less damaging to delicate plants than the blotting method. Errors in fresh weight determination between workers are also eliminated. Repeated centrifugation of plants, at 3-day intervals, was no more deleterious to their growth than the blotting method.     

One-hour downward alkaline capillary transfer for blotting of DNA and RNA.

The downward alkaline capillary transfer of DNA and RNA from agarose gel to a hybridization membrane was performed using a transfer solution containing 3 M NaCl and 8 mM NaOH. Under mild alkaline conditions, DNA and RNA were completely eluted from the agarose gel and bound to a hybridization membrane within 1 h. On the basis of this new method of transfer a blotting protocol, downward alkaline blotting, was elaborated. It provides a fast and efficient alternative to commonly used Southern and Northern blotting protocols. The downward alkaline blotting of DNA and RNA can be completed in 2.5 and 1.5 h, respectively, and can be used with both plastic and nitrocellulose membranes. In addition, the downward alkaline blotting protocol allows for a hybridization efficiency of DNA and RNA higher than that of the standard blotting protocols performed at neutral pH.     

Tips on improving the efficiency of electrotransfer of target proteins from Phos‐tag SDS‐PAGE gel

The sensitivity of Western blotting analysis after Phos‐tag SDS‐PAGE is occasionally inferior to that after normal (Phos‐tag‐free) SDS‐PAGE under similar experimental conditions, possibly as a result of inefficient electrotransfer from the Phos‐tag gel to the blotting membrane. We therefore present tips on improving the efficiency of electrotransfer of proteins in semidry and wet‐tank blotting. When model samples containing several standard phosphoproteins were subjected to semidry blotting, their electrotransfer efficiencies after Phos‐tag SDS‐PAGE were markedly inferior to those of their dephosphorylated counterparts in the same gel. This was ameliorated by immersing the electrophoresed Phos‐tag gel in a transfer buffer containing 1 mM EDTA for 30 min before electroblotting. Similarly, phosphoproteomes in crude cell extracts were inefficiently transferred by semidry blotting, but the efficiencies of their electrotransfer were improved by pretreatment with EDTA. In contrast, the efficiencies of wet‐tank blotting of the same samples were not dependent on the degree of phosphorylation, and the efficiencies of electrotransfer of all proteins from Phos‐tag gels were similar to those from normal gels. In some cases involving the use of a Phos‐tag gel, addition of 0.1% w/v of SDS to the transfer buffer significantly improved the electrotransfer.     

Use of a Western blotting technique in the purification of a cysteine proteinase inhibitor

In Western blotting procedures, proteins are resolved in sodium dodecyl sulfate-polyacrylamide gels with subsequent electrophoretic transfer onto nitrocellulose membranes. Although this procedure is generally employed as an analytical technique for assessing interactions of proteins with antibodies, the present report describes the use of Western blotting as a preparative procedure in the purification of a biologically active proteinase inhibitor from the cellular slime mold, Dictyostelium discoideum. The feasibility of using Western blotting for inhibitor purification depended upon the unique stability properties of the inhibitor under denaturing conditions.     

Detection of N-myc amplification in neuroblastomas using Southern blotting on fine needle aspirates

OBJECTIVE: To verify the possibility of detecting N-myc amplification in fine needle aspiration (FNA) cytology from neuroblastomas by the Southern blotting technique. STUDY DESIGN: Fifteen neuroblastomas diagnosed by FNA in the Department of Pathology, Hospital de S. Jo?o, between 1990 and 1998, were studied for N-myc amplification using the Southern blotting technique in cytologic and histologic material. RESULTS: DNA extraction from the cytologic material was performed in all cases (N = 15). In two cases the quality/quantity of the DNA did not allow the study of N-myc status by the Southern blotting technique. We detected N-myc amplification in 1 of 13 patients (7.6%) from whom material was available for genetic study. CONCLUSION: It is possible to use the Southern blotting technique to demonstrate N-myc amplification in material obtained from FNA of neuroblastomas.     

RNA干扰抑制结肠癌细胞血管内皮生长因子表达

目的 应用RNA干扰技术抑制结肠癌血管内皮生长因子（VEGF）表达。方法 将VEGF基因作为RNA干扰的靶区,通过E-RNAi网上提供的服务,设计两个特异的RNA干扰序列,将其装入含U6启动子的载体上,构建成抗VEGF基因的小发夹样RNA（shRNA）表达载体,再转染人结肠癌细胞HT29,通过RT-PCR、Northern杂交、免疫荧光和Western杂交,观察VEGF表达受抑的程度。结果 成功构建了两种抗VEGF基因的shRNA表达载体,RT-PCR、Northern杂交、免疫荧光和Western杂交,均发现其能明显抑制HT29细胞VEGF基因的表达,抑制率分别达42%、88%、73%和82%。结论 针对VEGF基因的shRNA表达载体能够明显抑制结肠癌细胞VEGF基因的表达。     

Evaluation of thymidylate synthase protein expression by Western blotting and immunohistochemistry on human colon carcinoma xenografts in nude mice.

In this study we investigated the relationship between thymidylate synthase (TS) protein expression, evaluated by Western blotting analysis and by immunohistochemistry (IHC), and growth rate in human colon xenograft tumors in nude mice. Human colon cancer cell lines were used to induce xenograft tumors and the tumor mass growth rate was calculated by measuring tumor size variations over time. TS 106 monoclonal antibody was used for both Western blotting and IHC TS detection. Tumor cell growth fraction was measured by Ki67/MIB1 immunolabeling and tumor cell growth rate by evaluating the mean nucleolar size in silver-stained sections. TS Western blotting values were related to tumor mass growth rate (p<0.001) and cell growth rate (p=0.002) but not to cell growth fraction (p=0.676). The degree of the IHC staining showed only a trend to be associated with TS protein expression measured on Western blotting, and was not related either to tumor mass growth or cell proliferation rate. Tumor xenografts were also characterized for TS promoter tandem repeat and p53 status. No relationship was observed between these variables and TS expression evaluated by both Western blotting and IHC analysis. Our results demonstrate that TS expression evaluated by Western blotting analysis is directly related to the tumor mass growth rate and question the use of the IHC approach to obtain precise quantitative information on TS expression in tumor samples.     

Enhancement of luminol-based immunodot and Western blotting assays by iodophenol

The application of 4-iodophenol as a signal enhancer for luminol-based immunodot and Western blotting assays was investigated. Immunodetection of biotin-bovine serum albumin (BSA) using a rat anti-biotin antibody for immunodot binding and goat anti-biotin peroxidase conjugate for Western blotting assays was employed to demonstrate the signal enhancement attainable with 4-iodophenol. The addition of 4-iodophenol at a final working concentration of 52.2 microM to a light-emitting substrate, luminol, resulted in 3-fold and 10-fold signal enhancements for Western blotting and immunodot binding detection of biotin-BSA, respectively.     

Utility of a direct dual-mode development analysis on blotted protein mixtures

Classic blotting methods remain a commonly applied approach to specific protein identification in gel electrophoresis of complex mixtures despite the inherent difficulty in band or spot matching due to significant variability of protein migration or localization in replicate blotting experiments. A direct application of both protein stain and protein blotting on a single membrane significantly reduces the complexity of the experiment and provides increased confidence of signal matching. Digital alignment of images acquired from both total protein stain and blotting development modes on a single membrane allows unambiguous spot or band assignments in these experiments as well as retention of quantitative information acquired from both modes of signal generation. A direct and simple method applying a fluorescent protein stain that is compatible with subsequent detection by antibody or lectin recognition factors along with common image adjustment software is examined. The utility of this blot dual-mode development method for direct protein recognition and quantification in one- and two-dimensional electrophoresis is demonstrated for bioanalytical objectives where replicate experiments are challenged by sample complexity.     

Specific monoclonal antibodies raised against Taura syndrome virus (TSV) capsid protein VP3 detect TSV in single and dual infections with white spot syndrome virus (WSSV)

The gene sequence encoding VP3 capsid protein of Taura syndrome virus (TSV) was cloned into pGEX-6P-1 expression vector and transformed into Escherichia coli BL21. After induction, recombinant GST-VP3 (rVP3) fusion protein was obtained and further purified by electro-elution before use in immunizing Swiss mice for production of monoclonal antibodies (MAb). One MAb specific to glutathione-S-transferase (GST) and 6 MAb specific to VP3 were selected using dot blotting and Western blotting. MAb specific to VP3 could be used to detect natural TSV infections in farmed whiteleg shrimp Penaeus vannamei by dot blotting and Western blotting, without cross reaction to shrimp tissues or other shrimp viruses, such as white spot syndrome virus (WSSV), yellow head virus (YHV), monodon baculovirus (MBV) and hepatopancreatic parvovirus (HPV). These MAb were also used together with those specific for WSSV to successfully detect TSV and WSSV in dual infections in farmed P. vannamei.     

Direct matrix-assisted laser desorption/ionization time-of-flight mass spectrometric identification of proteins on membrane detected by Western blotting and lectin blotting

We have developed new procedures to identify proteins after they are detected by Western blotting or other interactions such as lectin blotting on membranes. Our method is based on the combination of on-membrane MALDI-TOF mass spectrometry with piezoelectric chemical inkjet technology. Using this method the GroEL, FtsZ, DnaK, and GroES proteins were successfully identified from Escherichia coli after separation on two-dimensional gels, immunostaining, and on-membrane digestion. A glycoprotein detected by lectin blotting with concanavalin A was also identified using this technique.     

Protein purification and analysis: next generation Western blotting techniques

Introduction: Western blotting is one of the most commonly used techniques in molecular biology and proteomics. Since western blotting is a multistep protocol, variations and errors can occur at any step reducing the reliability and reproducibility of this technique. Recent reports suggest that a few key steps, such as the sample preparation method, the amount and source of primary antibody used, as well as the normalization method utilized, are critical for reproducible western blot results.

Areas covered: In this review, improvements in different areas of western blotting, including protein transfer and antibody validation, are summarized. The review discusses the most advanced western blotting techniques available and highlights the relationship between next generation western blotting techniques and its clinical relevance.

Expert commentary: Over the last decade significant improvements have been made in creating more sensitive, automated, and advanced techniques by optimizing various aspects of the western blot protocol. New methods such as single cell-resolution western blot, capillary electrophoresis, DigiWest, automated microfluid western blotting and microchip electrophoresis have all been developed to reduce potential problems associated with the western blotting technique. Innovative developments in instrumentation and increased sensitivity for western blots offer novel possibilities for increasing the clinical implications of western blot.     

Vacuum blotting enhances nucleic acid transfer

Nucleic acids can be transferred from an agarose get to a transfer membrane using capillary Southern blotting or electroblotting techniques. Vacuum blotting represents an alternative method that offers a number of advantages.     

Genetically fused protein A-luciferase for immunological blotting analyses

The gene expression plasmid pMALU5 for the fusion protein of protein A (SpA) with a complete sequence of firefly luciferase (Luc) was constructed. The fused gene was expressed in Escherichia coli, and the resulting SpA-Luc fusion protein was purified by one-step affinity chromatography on IgG-Sepharose. The protein retained both activities: IgG binding capability of protein A and enzymatic activity of luciferase. Blotting analyses were performed with the fusion protein to determine a tumor marker of alpha-fetoprotein (AFP). AFP was detected at the lowest detection limit of 5 pg by dot blotting and Western blotting. The SpA-Luc fusion protein provides a highly selective, sensitive, and versatile marker for blotting analyses.