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.     

CCA paper: a new two-dimensional cyanuric chloride-activated matrix for universal application in molecular biology

A novel two-dimensional cyanuric chloride-activated (CCA) paper has been developed. It is composed of a cellulosic base, covalently bound cyanuric chloride, and microprecipitated complex cyanuric chloride-sodium chloride crystals on its surface. CCA paper covalently binds nucleic acids and proteins. Its binding capacity for nucleic acids is about 400 micrograms/cm2. Sealed into nitrogen-filled bags and stored at -20 degrees C, it retains its binding activity for at least a year and is always ready for use. CCA paper has been successfully used for capillary and electroblotting of DNA, RNA, and proteins (Southern, Northern, and Western blotting) as well as for dot tests. Furthermore, it was applied to colony and plaque hybridization. A unique property of it is that it permits the staining of proteins after blotting and subsequent performance of radioimmunological detection of specific protein components. This has proven advantageous in two-dimensional Western blotting experiments. Of further importance is its ability to bind DNA fragments from one up to several hundred bp from polyacrylamide sequencing gels.     

Chemical cross-linking of Sm and RNP antigenic proteins

Nuclear extracts, competent for in vitro premessenger RNA splicing, were chemically cross-linked with thiol-reversible reagents in order to study the organization of proteins within ribonucleoprotein particles (RNPs) containing uridine-rich small nuclear RNAs (UsnRNPs). The distribution of select UsnRNP antigens within cross-linked complexes was determined by Western blotting of diagonal two-dimensional gels. On the basis of calculations from the molecular weights of cross-linked complexes containing UsnRNP common proteins B', B, and D, it is proposed that each of these proteins was associated with UsnRNP common proteins E and G. In addition, D' is proposed to be positioned close to D. The spatial distribution of UsnRNP common proteins was such that B' and B could not be cross-linked to D. The data also suggested that the 63-kDa U1 snRNP specific protein was cross-linked to other U1-specific proteins, particularly C, but not to the UsnRNP common proteins. We propose that part of the UsnRNP core of common proteins contains at least two asymmetrical copies of B':B:D:D':E:G with stoichiometries of 2:1:1:1:1:1 and 1:2:1:1:1:1.     

Proteomic analysis of human biopsy samples by single two-dimensional electrophoresis: Coomassie,silver, mass spectrometry,and Western blotting

Proteomic analysis of myocardial tissue from patient populations is critical to our understanding of cardiac disease, but has been limited until now by the small size of biopsies (approximately 20-50 microg), and complicated by the difference in relative abundance of contractile proteins over other cellular components. Here we describe an approach to analysis of myocardial biopsies from patients undergoing coronary artery bypass surgery. First, individual biopsies are selectively extracted, producing subfractions that correspond to the contractile proteins and the cytosolic proteins. Two-dimensional electrophoresis separated proteins are detected by first staining with Coomassie blue then silver, to permit a wider range of accurate quantification. Western blotting of two-dimensional separated samples, to validate peptide mass fingerprinting data, previously required additional gel separations for transfer since staining protocols are not compatible with transfer to membranes or immunoblotting. An existing silver destaining protocol was adapted to allow removal of silver from a whole gel, followed by transfer and Western blotting. An existing Coomassie blue removal protocol was also adapted to permit Western blotting of gels stained with Coomassie blue and silver. Together, these techniques permit peptide mass fingerprinting concurrent with Western blotting of a single protein spot from a single biopsy, eliminating the need for repeated gel separations, and improving spot alignment between immunoblots and stained gels. In the end, this approach may allow a more complete characterization of protein changes in small human biopsies, and also reduce the number of repeated gel separations necessary for a standard proteomic analysis.     

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.     

Use of the Mycoplasma hominis 102-116 kD proteins as antigen in an enzyme-linked immunosorbent assay

Mycoplasma hominis surface structures involved in human immune response and in the pathogenesis of this bacterial infection are inadequately defined. Attempts have been made to identify M. hominis surface proteins, to determine the antigenicity of these polypeptides, and to examine antigens which could lead to the development of species-specific diagnostic tests. By means of Western blotting, using a pool of sera from patients with culturally proven vaginal infection, most antigens recognized were surface exposed. Among these proteins, antigens of molecular weights between 102 and 116 kD were most consistently revealed. These polypeptides were recovered by electroelution and assayed in an IgG-ELISA. The electroeluted antigen specificity was examined by ELISA and immunoblotting with different mycoplasma species. Electroeluted proteins may be effective and specific for establishing a reliable diagnosis test.     

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.     

Fine specificities of autoantibodies directed against the Ro, La, Sm, RNP, and Jo-1 proteins defined by two-dimensional gel electrophoresis and immunoblotting

Although useful for specific purposes, immunofluorescence, precipitation in agarose gels, and the m.w. estimation of RNA or proteins immunoprecipitated from transformed cells often provide partial or ambiguous definition of autoantibody specificity. We have analyzed organ and cell extracts by one-and two-dimensional electrophoresis together with Western blotting to define the fine specificities of antibodies to the ribonucleoprotein (RNP) antigens Ro, La, Sm, RNP and Jo-1. One-dimensional analysis identified the Ro protein as a 57 kilodalton (kd) protein, although many anti-Ro sera also react with a 50 kd protein. La antisera react with 50 and 43 kd proteins. The 50 kd La protein readily breaks down into 43, 25, and smaller immunoreactive cleavage products. Partial proteolysis of Ro and La proteins in human spleen extracts produces similar immunoreactive products, providing evidence for a common structure. The major immunoreactive Sm antigens defined by human polyclonal antisera and a mouse monoclonal antiserum were doublets of 25/26 and 16/18 kd, whereas anti-RNP sera reacted with a protein of 68 kd. Most Sm-RNP antisera contained antibodies reactive with additional proteins, especially when whole cell extracts were used as a source of antigens. Two-dimensional analysis provided characteristic maps of the antigens. Ro and La were acidic, and La showed a unique set of acidic charge isomers at 50 and 43 kd. Anti-Sm antibodies reacted with discrete dots corresponding to both the acidic and basic regions of the first-dimension (charge) gels, whereas the RNP antigen showed a series of basic charge isomers of 68 kd. Many anti-Sm-RNP sera reacted with other closely spaced proteins of a similar charge and size to the Sm and RNP antigens, suggesting antibody cross-reactivity or reactivity with closely related functional proteins. Although Jo-1 had the same m.w. as the undegraded La antigen, the fingerprints were quite distinctive on two-dimensional electrophoresis. The results of this study indicate how the source and preparation of antigen extracts, as well as protein degradation, influence the m.w. determinations of soluble protein antigens. With these factors taken into account, two-dimensional fractionation with immunoblotting provides a highly discriminating, sensitive, and reproducible method of analysis of autoantibody specificity. This technique can be used to standardize reference antisera and to study protein antigens in normal and abnormal cell and tissue extracts, and could lead to new or more precise correlations with clinical disease.     

Detection of lipopolysaccharides in polyacrylamide gels by transfer to nitrocellulose followed by immunoautoradiography with antibody and 125I-protein A: "LPS blotting"

Lipopolysaccharides (LPS), which constitute the somatic (O) antigen of gram-negative bacteria, were used to demonstrate the procedure of LPS blotting involving the electrophoretic transfer of electrophoretically resolved LPS from sodium dodecyl sulfate-polyacrylamide gels to nitrocellulose filters. Immobilized LPS could then be immunoautoradiographically visualized in situ by reaction with specific anti-LPS antibody and subsequent binding of radioiodinated Staphylococcus protein A. LPS blotting is expected to provide an efficient and specific means of investigating the LPS (O) antigens of gram-negative bacteria.     

Proteins and antigens of merozoites and sporozoites of Eimeria bovis (Apicomplexa)

Proteins and antigens of first-generation merozoites and sporozoites of Eimeria bovis were examined using standard sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), Western blotting and lactoperoxidase iodination procedures. SDS-PAGE gels revealed both common and unique protein bands in merozoite and sporozoite extracts, ranging in molecular weight (Mr) from 15,000 to 215,000. Nitrocellulose immunoblots of separated proteins, when probed with sera obtained from immunized calves, revealed numerous IgG-binding antigens of Mr 18,000 to 180,000 in merozoites and Mr 28,000 to approximately 118,000 in sporozoites. Although merozoite and sporozoite preparations each contained antigens of different molecular weights, 4 antigens had the same migratory distance in both preparations (Mr 58,000, 70,000, 83,000, 98,000). Of 3 types of immune sera used to probe immunoblots, serum taken from a calf that had been inoculated with oocysts of E. bovis and boosted 10 wk later by subcutaneous injection with 2 X 10(7) live merozoites emulsified in Freund's complete adjuvant consistently identified and reacted more intensely with more antigens of merozoites and sporozoites than the other immune sera tested. Autoradiographic analysis of radioiodinated parasites revealed major surface proteins on merozoites of between 15,000 and 18,000 Mr and 3 surface proteins on sporozoites of Mr 28,000, 77,000, and 183,000. All but the 183,000 protein elicited an IgG antibody response in the host.     

Immunoproteomic assay of secreted proteins of <Emphasis Type="Italic">Streptococcus suis</Emphasis> serotype 9 with convalescent sera from pigs

Streptococcus suis is an important pathogen of pigs. In China, in addition to S. suis serotype 2, S. suis serotype 9 (SS9) is also a prevalent serotype. There is no vaccine available for SS9. An immunoproteome-based approach was developed to identify SS9 immunogenic proteins for vaccine development. Secreted proteins extracted from SS9 strain GZ0565 were screened by two-dimensional Western blotting using convalescent sera from pigs. Protein spots were excised from preparative gels and were identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry, which led to the identification of ten immunogenic proteins (sortases, ABC transporter substrate-binding protein–maltose/maltodextrin, ABC transporter periplasmic protein, CHAP domain containing protein, peptidoglycan-binding LysM, elongation factor Tu, elongation factor G, thymidine kinase, molecular chaperone DnaK, hypothetical protein SSU98_2184). These novel immunogenic proteins, which are encoded by genes that are reasonably conserved among SS9 strains, may be developed as antigens for further study of SS9 vaccine.     

Zinc-binding proteins detected by protein blotting

The Western blotting technique was used for the detection of zinc-binding proteins. Proteins were separated electrophoretically on 15% polyacrylamide-sodium dodecyl sulfate minigels, the gels were soaked in a reduction buffer, and the proteins were transferred to nitrocellulose filters. Zinc-binding proteins were probed with radioactive zinc (65Zn) and were detected by autoradiography. This technique allows the detection of as little as 20 to 100 pmol of zinc metalloproteins.     

人肺鳞癌组织的血清蛋白质组学的比较分析

采用以肿瘤免疫学与蛋白质组学(proteomics)研究技术有机地结合为基础的血清蛋白质组学研究体系(serologicproteomeanalysis ,SERPA)筛选肺癌分子标志物.对10例人肺鳞癌组织,应用双向凝胶电泳(two dimensionalelectrophoresis ,2 DE)技术对同一肺鳞癌组织的细胞总蛋白同时进行电泳后获得3张相同的凝胶,其中一块2 DE凝胶经银染显色作为平行胶,其余两块2 DE凝胶经电转膜将凝胶中的蛋白质转至硝酸纤维素(NC)膜上,然后分别与肺癌患者的自身血清以及正常对照血清进行Western印迹分析,获取Western印迹反应图谱.经计算机图像分析识别差异反应的蛋白质,然后与平行胶比较找出相应的差异反应蛋白质点.获得了分辨率较高的人肺鳞癌组织与患者的自身血清以及正常对照血清的Western印迹反应图谱;图像分析共识别36±8个差异反应的蛋白质;在平行胶上找到了匹配的差异反应蛋白质点.对2 0个差异蛋白质点进行了肽质指纹图分析,鉴定出14个与细胞生长增殖、细胞代谢、细胞周期调控、信号转导等有关的肺鳞癌相关抗原.通过血清蛋白质组技术对肺鳞癌组织进行的研究,建立了分辨率较高的人肺鳞癌组织与患者的自身血清以及正常血清的Western印迹反应图谱,成功鉴定14个肺鳞癌相关抗原,为进一步筛选用于肺鳞癌诊断、治疗和预后评估     

弗氏志贺菌磷酸化蛋白的检测

目的:检测弗氏志贺菌全菌蛋白中被磷酸化修饰的蛋白。方法:制取弗氏2a志贺菌2457T野生株全菌磷酸化蛋白样品时加入磷酸化酶抑制剂,随后对样品进行双向电泳,以抗磷酸丝氨酸/苏氨酸/酪氨酸抗体为免疫探针,通过Western印迹找到被磷酸化的蛋白,并进行胶内酶解及MALDI-TOF质谱分析。结果与结论:共检测到13个磷酸化蛋白,其中9个为代谢途径中的酶。     

Applying phage antibodies to proteomics: selecting single chain Fv antibodies to antigens blotted on nitrocellulose

Two-dimensional gel electrophoresis is a powerful tool for identification of proteins that differ between patients with qualitatively or quantitatively different disease states. Further characterization of these protein differences would be greatly facilitated by the availability of antibodies that could be used to detect and quantitate the temporo-spatial pattern and cellular and tissue location of the different proteins. To generate such antibodies, methods were developed which permit the successful selection of monoclonal phage antibodies from phage display libraries against antigens blotted from SDS-PAGE gels onto nitrocellulose. First, it was determined that nitrocellulose and PVDF membranes gave significantly lower levels of background phage binding than two other membranes studied. Next, it was determined that blocking with fish gelatin and binding in the presence of 0.5 M NaCl could reduce nonspecific binding 10,000-fold and result in enrichment ratios greater than 500-fold with antigen concentrations as low as 1 ng/mm(2). When optimized conditions were applied to phage antibody libraries, panels of monoclonal phage antibodies were generated against the proteins ErbB2 and bovine serum albumin electroblotted from SDS-PAGE gels onto nitrocellulose. Antibodies were obtained with as little as 10 to 1 ng of antigen, depending on whether the libraries displayed single or multiple copies of antibody per phage. The antibodies worked as reagents in both ELISA and Western blotting.     

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.     

Detection of DNA-protein binding in western blots by phosphorus-labeled and biotinylated DNA probes

Eukaryotic DNA-binding proteins can be detected by a filter binding assay combining protein blotting on nitrocellulose, incubation with DNA by filtration, and the application of radioactively or nonradioactively labeled DNA probes. Basic nuclear and non-nuclear standard proteins are assayed in dot blots as well as in Western blots from sodium dodecyl sulfate gels. The DNA-binding ability of fractionated proteins is compared employing two different blotting techniques, conventional electro-transfer and protein-renaturating capillary transfer. Biotinylated DNA probes exhibit high sensitivity and a distinct discrimination of detection signals corresponding only to defined DNA-binding proteins. In contrast, phosphorus-labeled DNA probes show higher sensitivity, but less effective resolving power, especially for bands localized close to each other. Using the DNA-incubation procedure described, biotinylated DNA probes are preferable to radioactively-labeled probes for screening DNA-binding proteins in complex protein fractions.     

Identification and characterization of GroEL and DnaK homologues in Thiobacillus ferrooxidans

The major heat shock proteins from Thiobacillus ferrooxidans were identified as DnaK and GroEL equivalents by Western blotting and analysis of the N-terminal amino acid sequence of spots isolated from dried 2-D polyacrylamide electrophoresis gels. The T. ferrooxidans chaperonins showed 70% and 80% identity with the Escherichia coli GroEL and DnaK, respectively. By using electrophoresis with a transverse pore gradient of cross-linked polyacrylamide and nondenaturing conditions followed by Western blotting, we found that the GroEL proteins from both bacteria formed a 14-mer, whereas E. coli DnaK protein existed partially as a dimer and the T. ferrooxidans DnaK-equivalent showed only a monomeric nature under our experimental conditions.     

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.