Luminescent immunodetection of western-blotted proteins from coomassie-stained polyacrylamide gel

Immunodetection with horseradish peroxidase-linked antibodies on Coomassie-stained nitrocellulose blots can be performed efficiently and rapidly with the peroxidase substrate luminol. The luminescence produced is detected with radioautographic film. This procedure allows a direct identification of immunodetected bands of stained nitrocellulose sheets without using radiolabeled secondary antibodies. Because of its convenience and sensitivity, this method could be particularly suitable for purification of immunodetected proteins.     

用硝酸纤维素膜亲和法纯化抗体

介绍一种改进的纯化抗体方法。将抗原蛋白固定在硝酸纤维素膜上,然后对抗体进行亲和纯化,能快速有效获得高特异性抗体。     

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.     

A rapid procedure for preparing fluorescein-labeled specific antibodies from whole antiserum: its use in analyzing cytoskeletal architecture

A rapid method for the direct conjugation of affinity-purified antibodies with fluorescein (termed DCAPA) is described. This procedure involves the immobilization of antibodies as antigen-antibody complexes on nitrocellulose blots, and subsequently the bound antibodies are reacted with fluorescein isothiocyanate. An enriched sample of smooth muscle tropomysin transferred to nitrocellulose paper by the Western blotting procedure has been used as the affinity medium for purification of specific tropomyosin antibody from whole rabbit antiserum. Direct conjugation of the antibody with fluorescein was carried out following the binding of antibody to antigen. Direct conjugation and affinity purification of antibodies directed against tropomyosin was accomplished in 2-3 d using an enriched tropomyosin sample and whole antiserum directed against tropomyosin. The immunofluorescence images obtained with this procedure exhibit distinct advantages with regard to background fluorescence and overall specificity of antibody binding. The usefulness of this direct conjugation method in various experimental protocols is discussed.     

A dot-blot assay for the low density lipoprotein receptor

We describe a new method for detecting the interaction of low density lipoprotein with its receptor using unmodified nitrocellulose as support for membrane protein. The method is specific and sensitive down to 3 micrograms of membrane protein. Unlabeled LDL, but not HDL, competes with 125I-labeled LDL for binding, and binding is abolished by pretreatment of the membranes with pronase and is dependent upon the presence of Ca2+. Furthermore, modification of arginine or lysine residues on LDL abolishes the lipoprotein interaction with the receptor protein supported on the nitrocellulose. When the membranes are solubilized with octyl glucoside, purification steps of the receptor can be directly followed with no interference of the detergent, therefore eliminating the need for its removal. The increased expression of LDL receptors on liver membranes from estradiol-treated rats was also demonstrated. We suggest, therefore, that this method can be used to detect the presence of LDL receptors on minute amounts of membrane protein.     

A method for the production of highly specific polyclonal antibodies

Two polyclonal antibodies directed against paramyosin and tropomyosin from Owenia fusiformis (a marine polychete annelid) were obtained using a new method of immunization. After purification by two-dimensional gel electrophoresis, proteins were transferred onto a nitrocellulose sheet using the Western blot technique. The proteins bound to their cellulose support were injected into rabbits without Freund's adjuvant and without solubilization of nitrocellulose with dimethyl sulfoxide. Highly specific polyclonal antibodies were generated.     

A novel probe Au(III) for chemiluminescent image detection of protein blots on nitrocellulose membranes

A new method, based on the chloroauric acid-enhanced luminol chemiluminescence, is established for the chemiluminescent imaging detection of protein blots on nitrocellulose membranes. After transferring to the nitrocellulose (NC) membranes, various proteins in human serum can be easily detected using this method. Simplicity and wide applicability are achieved, without the need of expensive antibodies or tedious immunoassay procedures. Furthermore, neither noxious materials nor radioactive pollution is produced. The successful detection of proteins is due to the binding of Au(III) to the protein blots and the chemiluminescent character of the enhanced luminol signal. As a novel chemiluminescent detection method, it offers significant biological analytical potentials in biochemistry and in molecular biology.     

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.     

Visualization of antigen on nitrocellulose membrane by the oxidative coupling reaction of N,N'-dimethyl-p-phenylenediamine and 4-chloro-1-naphthol

A method which allows the highly sensitive and simple immunodetection of antigen-antibody complexes on nitrocellulose papers has been developed. The method is a modification of the procedure known as the "Nadi reaction" (oxidative coupling of 1-naphthol and N,N'-dimethyl-p-phenylendiamine) for histochemical purposes. Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrophoretically transferred to nitrocellulose membranes. Bound proteins were first reacted with a primary antibody and then with a horseradish peroxidase-labeled second antibody. The antigen-antibody complexes on the membranes were visualized with the oxidative coupling solution containing N,N'-dimethyl-p-phenylendiamine and 4-chloro-1-naphthol. Sensitivity was enhanced 4 to 16 times by the new method relative to that of the 4-chloro-1-naphthol method or the 3,3'-diaminobenzidine method.     

Quantitation of mucus glycoproteins blotted onto nitrocellulose membranes

A sensitive assay for mucus glycoproteins (mucins) and fragments thereof is presented. The macromolecules are blotted onto nitrocellulose membranes and visualized using a periodate-Schiff (PAS) reaction and the color yield quantitated with an image analysis system used as a reflectance densitometer. At least 50 ng of the macromolecules was detected. "Whole" mucins and subunits were assayed on 0.2-micron pore size nitrocellulose membranes whereas immobilization of the high-molecular-weight mucin glycopeptides (Mr 300-500,000) required pretreatment of membranes with poly-L-lysine. Binding of the glycopeptides to the polylysine-treated membranes was found to decrease with increasing salt concentration suggesting an electrostatic interaction. The data obtained with this method and a solution PAS assay are in good agreement but the former is more sensitive and can be performed on samples dissolved in chaotropic solvents.     

Viral immunoblotting: A sensitive method for detecting viral-specific oliogoclonal bands in unconcentrated cerebrospinal fluid

A new method for detecting viral antibodies in cerebrospinal fluid is described. The technique has many advantages over previously published methods in that it is highly sensitive eliminating the need to concentrate the CSF, takes 5 h to complete, avoids the use of radionucleides, and most importantly circumvents problems associated with prozone effects which occur in immunoprecipitation reaction since the viral antigen is immobilized on nitrocellulose membranes.     

Purification of outer membrane proteins of the gram-negative bacterium Neisseria gonorrhoeae

A system of protein purification, using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electroblotting, that results in purified outer membrane proteins of the gram-negative bacterium Neisseria gonorrhoeae is described. The proteins, which ranged in apparent molecular mass from approximately 31,000 to approximately 92,000 Da, were located by naphthol blue black staining, eluted from nitrocellulose membranes using 88% formic acid, and precipitated by the addition of concentrated ammonium hydroxide. Up to 65% of the original protein present was recovered by this procedure. The resultant purified protein could then be resuspended in aqueous buffer by brief sonication, making it available for further structural and in vivo immunological analyses. Proteins purified in this manner retain their original antigenicity when probed with polyclonal and monoclonal antibodies, and are structurally unaltered by the purification process. This procedure makes it possible to acquire easily usable quantities of highly insoluble outer membrane proteins of gram-negative bacteria.     

Quantification of DNA-dependent RNA polymerase subunits and initiation factor(s) by antibody-linked polymerase assays

The antibody-linked polymerase assay is a method which allows one to assign RNA polymerase activity to SDS-denatured polypeptides on nitrocellulose membranes using antibodies which were raised against only partially purified polymerase preparations. Here we show that with this method not only enzyme subunits but also initiation factor(s) can be determined in crude homogenates. Moreover the determination is quantitative. Therefore changes in the amount of individual polymerase subunits and factor(s) can be visualized within different crude homogenates.     

Development of a dot-blot assay for screening monoclonal antibodies to low-molecular-mass drugs

Dot-blot is a versatile and simple analysis to perform. We adapted this method as a simple identity test for monoclonal antibodies to a number of small compounds: three transplant drugs, an anticonvulsant, a steroid, an anticancer drug, and an antibiotic. Immunology-based identity tests using low-molecular-mass organic compounds have historically been a challenge to develop. We modified the traditional dot-blot assay to serve as an identity test for monoclonal antibodies to carbamazepine, sirolimus, tacrolimus, cyclosporine, cortisol, methotrexate, and gentamicin. The primary obstacle was the immobilization of these organic compounds on nitrocellulose as nitrocellulose is also soluble in most of the organic solvents in which the compounds are soluble. We evaluated different membranes, solvents, and chemical forms of these organic compounds to overcome this challenge. A number of incubation and washing solutions were also investigated. By varying the chemical form, concentration, and incubation conditions, a set of effective and reproducible identity tests were developed for these monoclonal antibodies.     

Improving the quality of immunoblots by chromatography of polyclonal antisera on keratin affinity columns

Unwanted reactivity of polyclonal antisera against keratins ("fingerprint proteins") is a problem commonly encountered when proteins transferred to nitrocellulose are studied by immunoblotting. Immunoreactivity against keratins is generally accompanied by a spotted background. This antikeratin immunoreactivity could be removed by adsorption of the antisera to human keratin bound to nitrocellulose. Larger amounts of antisera were purified from contaminant antikeratin antibodies by a single passage over a column of human keratin coupled to activated CH-Sepharose 4B. In contrast to nonpurified antisera and their IgG fractions, the column effluent no longer recognized the Mr 55,000-70,000 keratin proteins and exhibited a marked decrease in background labeling. We propose this simple method as a valuable alternative when affinity purification of polyclonal antisera on antigen columns is not practical.     

A note on colony-blot double-stain method for isolation of Vibrio cholerae serotype 01 from specimens heavily contaminated with non-01 Vibrio cholerae

A colony-blot double-stain method was developed to identify individual colonies of Vibrio cholerae serotype 01 (pandemic strain) in mixed bacterial cultures on solid media. The colonies are transferred from agar to nitrocellulose membranes for an enzyme-linked immunosorbent assay (ELISA). Colonies of 01 vibrios bind the enzyme-linked antibodies and appear as brown dots on the membranes; pale black dots develop at the site of replicated colonies of other bacteria as a result of the activity of endogenous oxidase-like enzymes and serve as reference points. The results indicate that the colony-blot double-stain method is useful for the isolation of colonies of V. cholerae serotype 01 in specimens that are heavily contaminated with non-01 vibrios.     

Immunodetection with streptavidin-acid phosphatase complex on Western blots

A technique for the detection of nanogram amounts of protein blotted onto nitrocellulose membranes has been developed using nonradioactive probes. Protein transferred to nitrocellulose membranes is detected by a specific antibody followed by incubation with biotinylated anti-antibody. After addition of streptavidin-acid phosphatase complex, incubation with fast violet B salt produces sharp magenta bands. This method allows detection of bands containing less than 20 ng of protein. The procedure does not use radioactive or carcinogenic materials.     

A refined method for silver staining of nucleic acids fixed on nitrocellulose

A refined silver staining method was developed to stain nucleic acids fixed onto nitrocellulose membranes and nylon-based membranes. Approximately 4 ng RNA or DNA can be stained with this method with no protein interference. This method involves simple repetition of immersions of membranes in three solutions prepared from common chemicals. The total staining time is less than 30 min.     

A rapid method for fibronectin purification on nitrocellulose membranes suitable for tissue culture

We have developed a simple method for plasma fibronectin purification based on the well-known gelatin binding property of fibronectin. In this procedure we immobilize the melted gelatin to nitrocellulose membranes; these are then used to affinity-purify the fibronectin from the plasma sample. The fibronectin is eluted from the membrane by treatment with 8 M urea. The procedure described here gives a yield of up to 60% (from presumed fibronectin concentration) and the fibronectin obtained is homogeneous in SDS-PAGE and biologically active, as assessed by a cell migration assay. The method is rapid, simple, inexpensive, does not require the use of chromatographic equipment and is suitable for tissue culture applications.     

Purification and analysis of mitochondrial membrane proteins on nondenaturing gradient polyacrylamide gels

A nondenaturing gradient polyacrylamide gel electrophoresis method is described for the resolution of membrane proteins. Bovine heart inner mitochondrial membranes were solubilized in Triton X-100 and individual complexes were identified by staining for activity and protein. Succinate dehydrogenase was isolated by band excision and shown by electrophoresis under denaturing conditions to be highly purified. In addition, the electrophoretic transfer of NADH dehydrogenase to nitrocellulose was demonstrated. The enzyme was identified on the resulting blot by activity staining and the binding of monospecific antibodies.