Reversible staining and peptide mapping of proteins transferred to nitrocellulose after separation by sodium dodecylsulfate-polyacrylamide gel electrophoresis

We describe a staining technique, using Ponceau S in very mild conditions, by which proteins can be visualized on nitrocellulose replicas without being permanently fixed to the membrane itself, thus allowing subsequent procedures such as immunoblotting or preparative elution of the proteins to be performed. This staining technique can detect 250 to 500 ng protein, which is essentially the same sensitivity seen for Coomassie blue staining of proteins on nitrocellulose. The Ponceau S staining technique was used to locate proteins on nitrocellulose replicas for subsequent in situ radioiodination and trypsin digestion, followed by separation of the resultant digests in two-dimensional peptide analysis. Staining proteins with Ponceau S did not interfere with either the radioiodination or trypsin digestion, as indicated by essentially identical peptide patterns being obtained for the internal protein p26 from equine infectious anemia virus, regardless of whether the digests were prepared from polyacrylamide gel slices or nitrocellulose sections. The combination of preparation of radioiodinated tryptic digests on nitrocellulose and subsequent two-dimensional analysis is sensitive enough to detect peptide additions and deletions occurring in the surface antigen gp90 recovered from two antigenically distinct strains of equine infectious anemia virus. Thus these procedures provide a relatively simple, inexpensive, and highly reproducible technique for the analysis of as little as 250 nanograms of protein after separation by electrophoresis in polyacrylamide gels.     

Immunodetection of biotinylated lymphocyte-surface proteins by enhanced chemiluminescence: a nonradioactive method for cell-surface protein analysis.

Biotinylation and radioiodination have been compared for labeling lymphocyte-surface proteins and the labeled proteins symmetrically immunoprecipitated with antibodies recognizing major lymphocyte markers such as the murine Thy-1, CD25 (the alpha subunit of the interleukin-2 receptor), CD45, and human CD2 glycoproteins. The detection of biotinylated proteins by enhanced chemiluminescence after transfer to nitrocellulose was found to be fast and as efficient as the detection of iodinated proteins by autoradiography. The vectoriality of cell-surface biotinylation was ascertained by two-dimensional electrophoresis of the cellular extract in which the major cytoplasmic proteins were not found biotinylated. This nonradioactive labeling procedure offers a convenient and efficient alternative to radiolabeling of cell surfaces for the biochemical analysis of extracellular domains of membrane proteins.     

A 39-kD plasma membrane protein (IP39) is an anchor for the unusual membrane skeleton of Euglena gracilis

The major integral plasma membrane protein (IP39) of Euglena gracilis was radiolabeled, peptide mapped, and dissected with proteases to identify cytoplasmic domains that bind and anchor proteins of the cell surface. When plasma membranes were radioiodinated and extracted with octyl glucoside, 98% of the extracted label was found in IP39 or the 68- and 110-kD oligomers of IP39. The octyl glucoside extracts were incubated with unlabeled cell surface proteins immobilized on nitrocellulose (overlays). Radiolabel from the membrane extract bound one (80 kD) of the two (80 and 86 kD) major membrane skeletal protein bands. Resolubilization of the bound label yielded a radiolabeled polypeptide identical in Mr to IP39. Intact plasma membranes were also digested with papain before or after radioiodination, thereby producing a cytoplasmically truncated IP39. The octyl glucoside extract of truncated IP39 no longer bound to the 80-kD membrane skeletal protein in the nitrocellulose overlays. EM of intact or trypsin digested plasma membranes incubated with membrane skeletal proteins under stringent conditions similar to those used in the nitrocellulose overlays revealed a partially reformed membrane skeletal layer. Little evidence of a membrane skeletal layer was found, however, when plasma membranes were predigested with papain before reassociation. A candidate 80-kD binding domain of IP39 has been tentatively identified as a peptide fragment that was present after trypsin digestion of plasma membranes, but was absent after papain digestion in two-dimensional peptide maps of IP39. Together, these data suggest that the unique peripheral membrane skeleton of Euglena binds to the plasma membrane through noncovalent interactions between the major 80-kD membrane skeletal protein and a small, papain sensitive cytoplasmic domain of IP39. Other (62, 51, and 25 kD) quantitatively minor peripheral proteins also interact with IP39 on the nitrocellulose overlays, and the possible significance of this binding is discussed.     

Selective radioiodination of the apical and luminal cell surfaces: in vitro and in situ experiments on vascular endothelial cells with Iodogen-coated Sephadex

A gentle and nonexpensive agent for selective radioiodination of the cell surface proteins was obtained by plating aliquots of Iodogen on dried Sephadex beads 50-60 microns in diameter. Iodogen-coated Sephadex inherits Iodogen properties: it is stable and virtually insoluble in water, allowing rapid iodination of the cell surface proteins in the solid phase with 125I-. Iodination is terminated by simply removing the beads. The agent was tested on bovine aortic endothelial cells in culture and on rabbit aortic endothelial cells in situ. Light and electron microscopic studies revealed that during radioiodination, apparently no ultrastructural modifications occurred in the endothelial cells. In addition, experiments with 51Cr (used as an indicator of endothelial cell injury) demonstrated that during iodination the cell integrity was preserved. The technique reported here may be generally applied for selective radioiodination of the apical surface proteins of various cultured cells and of the luminal endothelial surface of large blood vessels.     

Radioiodination and 125I-labeled peptide mapping of proteins on nitrocellulose membranes

A rapid procedure for generating dozens of 125I-labeled peptide maps from a protein band excised from a single lane of a sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel has been developed. Proteins, which can be rapidly purified by 2 X SDS-PAGE separation, are electroblotted onto nitrocellulose paper (NCP) and located by aqueous naphthol blue-black staining. All subsequent steps of radioiodination, and enzyme or chemical cleavage, are carried out on the NCP making it possible to test a variety of cleavage reagents on the same protein sample. The resultant peptidic residues, which can be separated by thin-layer electrophoresis-thin-layer chromatography (2D TLE-TLC), SDS-PAGE, or HPLC, can be used in comparative studies or they can be recovered for further structural and immunological analyses.     

Protein transfer from fixed, stained, and dried polyacrylamide gels and immunoblot with protein A-gold

The method of electrophoretically transferring proteins from fixed and stained polyacrylamide gels onto nitrocellulose paper has been reevaluated. It is shown that the tedious destaining of gels is not necessary because Coomassie brilliant blue, although it binds tenaciously to nitrocellulose paper, does not reduce the transfer efficiency of proteins. However, its presence impairs the visibility of proteins as detected, for instance, by the immunogold technique. Therefore, a rapid method for the complete removal of the stain from the nitrocellulose paper after completion of the immunogold procedure was developed. Furthermore, it is shown that proteins from dried polyacrylamide gels can still be transferred onto nitrocellulose sheets with an efficiency of approximately 50% compared to proteins transferred from fixed gels.     

Fluorescent labeling of nitrocellulose-bound proteins at the nanogram level without changes in immunoreactivity

Proteins blotted on nitrocellulose were stained with either 5-dimethylamino-1-naphthalene-sulfonylchloride (dansyl chloride) or fluorescein isothiocyanate. In both cases the staining procedure can be completed in less than 30 min. The sensitivity for detecting fluorescent-labeled proteins on nitrocellulose was 0.5 ng using a dot test. This was accomplished by transparentizing the nitrocellulose with either immersion oil or toluene. Dansylated proteins were successfully utilized for optimizing the electroblotting procedure. In the presence of 0.2% sodium dodecyl sulfate and 20% methanol the distribution of proteins on the nitrocellulose was an exact replica of the protein pattern seen in the polyacrylamide gel. The fluorescent labeling did not affect the antigenic properties of proteins allowing the subsequent probing with antisera. For this procedure, only one set of samples is needed to obtain accurate photographic records of the gel, the nitrocellulose blot, and the probed blot.     

Analysis of electroblotted proteins by mass spectrometry: protein identification after Western blotting

We describe a new approach for the identification and characterization by mass spectrometry of proteins that have been electroblotted onto nitrocellulose. Using this method (Blotting and Removal of Nitrocellulose (BARN)), proteins can be analyzed either as intact proteins for molecular weight determination or as peptides generated by on-membrane proteolysis. Acetone is used to dissolve the nitrocellulose and to precipitate the adsorbed proteins/peptides, thus removing the nitrocellulose which can interfere with MS analysis. This method offers improved protein coverage, especially for membrane proteins, such as uroplakins, because the extraction step after in-gel digestion is avoided. Moreover, removal of nitrocellulose from the sample solution allows sample analysis by both MALDI- and (LC) ESI-based mass spectrometers. Finally, we demonstrate the utility of BARN for the direct identification of soluble and membrane proteins after Western blotting, obtaining comparable or better results than with in-gel digestion.     

Radioiodination and I-labeled peptide mapping of proteins on nitrocellulose membranes

A rapid procedure for generating dozens of ¹²⁵I-labeled peptide maps from a protein band excised from a single lane of a sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel has been developed. Proteins, which can be rapidly purified by 2× SDS-PAGE separation, are electroblotted onto nitrocellulose paper (NCP) and located by aqueous naphthol blue-black staining. All subsequent steps of radioiodination, and enzyme or chemical cleavage, are carried out on the NCP making it possible to test a variety of cleavage reagents on the same protein sample. The resultant peptidic residues, which can be separated by thin-layer electrophoresis-thin-layer chromatography (2D TLE-TLC), SDS-PAGE, or HPLC, can be used in comparative studies or they can be recovered for further structural and immunological analyses.     

Antigenic complexity of Treponema pallidum: antigenicity and surface localization of major polypeptides

The protein structure of Treponema pallidum was characterized by two-dimensional electrophoresis (2DE), consisting of isoelectric focusing (IEF, pH 5 to 7) in the first dimension and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in the second dimension. Up to 85 major polypeptide species could be detected in the organisms in 2DE gels by Coomassie Blue staining. The antigenicity of the individual polypeptides was determined by transferring the 2DE pattern to nitrocellulose paper and utilizing a sensitive immunoperoxidase procedure to demonstrate the reactivity of immunoglobulins in sera obtained from rabbits infected intratesticularly at least 6 mo previously. The infected rabbit serum reacted with virtually every major polypeptide detectable by protein staining techniques, indicating that infected rabbits produce antibodies against nearly all major T. pallidum proteins at the time when the animals exhibit systemic resistance to reinfection. Surface radioiodination of freshly purified T. pallidum by an Iodogen procedure yielded preferential labeling of a major polypeptide with an apparent m.w. of 39,000. The results of this study indicate that the antigenic complexity of T. pallidum is much greater than described previously. The 39-kd polypeptide appears to be a major surface constituent of T. pallidum and as such may play an important role in the induction of immunity to syphilis.     

Optimal conditions for lactoperoxidase catalyzed radioiodination of external proteins on mouse erythrocytes

Optimal conditions were established for specific labelling of the surface proteins of mouse erythrocytes using lactoperoxidase-catalyzed radioiodination. The levels of H2O2 and I-, and cell concentrations required for restriction of haemoglobin labelling to less than 5% of the total 125I-protein, were different for radioiodination employing direct H2O2 addition or generation of H2O2 with glucose oxidase plus glucose. Preparation of mouse erythrocyte ghosts by hypotonic lysis caused loss of some minor labelled proteins present on intact cells and shifts to lower molecular weights of others. It is therefore important to solubilize labelled cells directly in electrophoresis buffer to avoid artifactual degradation of labelled proteins. The extent of labelling internal cell proteins was measured by a procedure suitable for the comparison of a large number of samples: solubilized radioiodinated erythrocytes were electrophoresed on 14% acrylamide gels and the radioactivity determined in the haemoglobin band which migrates separately from other proteins. The major labelled protein on the mouse erythrocytes had an apparent molecular weight of 92,000, and may be analogous to Band 3 of the human erythrocyte.     

Fluorescent monitoring of proteins during sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting

Fluorescent labeling of proteins was found to be a very sensitive and reliable alternative to conventional methods for monitoring proteins on Western blots. Proteins were labeled with 2-methoxy-2,4-diphenyl-3(2H)-furanone (MDPF) before SDS-PAGE. After electrophoresis and subsequent electro-blotting the fluorescent-labeled proteins were visible upon ultraviolet illumination of the nitrocellulose membranes, and could be photographed to yield an accurate record of the blots before subsequent serological analysis. The sensitivity for detecting MDPF-labeled proteins on nitrocellulose was 100-200 ng, 50 to 100 fold less sensitive than on gels. Fluorescent-labeled TMV and MStpV capsid proteins that were blotted onto nitrocellulose still reacted in serological tests and were detected when present in quantities as low as 100 pg. Fluorescent labeling allows accurate photographic records of the SDS-gel, blot and probed blot using only one sample, and no subsequent staining steps are required.     

Isolation of ribosomal protein-RNA complexes by nitrocellulose membrane filtration: equilibrium binding studies.

E. coli ribosomal proteins are retained by nitrocellulose filters. In contrast, 16S RNA passes through nitrocellulose filters. We have found that specific protein-RNA complexes involving single proteins also pass through nitrocellulose filters. Thus, by utilizing radioactively labeled r-proteins, nitrocellulose filtration can be used to study directly and sensitively the stoichiometry of r-protein-RNA association. The filtration process maintains near equilibrium conditions, making it applicable to weak as well as strong protein-RNA associations. We have used nitrocellulose filtration to obtain saturation binding curves for the association of proteins S4, S7, S8 and S20 with 16S RNA. In each case, the stoichiometry of binding was one mole of protein or less per mole of RNA. The stoichiometry of protein S8 binding to 16S RNA measured by filtration is comparable to that observed by sucrose gradient centrifugation. Association constants for the binding of proteins S4, S8 and S20 to 16S RNA have been determined by analysis of the saturation binding curves and were found to range from .3-6 X 10(7)M-1.     

Reaction of monoclonal antibodies with plasma membrane proteins after binding on nitrocellulose: renaturation of antigenic sites and reduction of nonspecific antibody binding

The immunochemical reaction of monoclonal antibodies directed against native membrane proteins was investigated after their separation in sodium dodecyl sulfate polyacrylamide gels and electrotransfer to nitrocellulose. Nonspecific binding of antibodies to membrane proteins, which was increased by beta-mercaptoethanol treatment or heat denaturation of the antibodies, could be significantly reduced if 1 M D-glucose plus 10% (v/v) glycerol was added during the incubation with the antibodies. It was found that specific antibody binding was drastically reduced by SDS treatment of the membrane proteins. During the electrotransfer to nitrocellulose and the simultaneous removal of SDS, some increase in antibody binding was observed. Considerable renaturation of antigenic sites in the blotted proteins could be induced if the nitrocellulose blots were incubated for 16 h at 37 degrees C in phosphate-buffered saline. With the introduction of both modifications, the renaturation step, and the addition of D-glucose and glycerol to reduce nonspecific antibody binding, the immunoblot technique may be successfully applied to detect conformational antibodies against membrane proteins.     

A two-step procedure for efficient electrotransfer of both high-molecular-weight (greater than 400,000) and low-molecular-weight (less than 20,000) proteins

We have developed conditions for the efficient electrotransfer from polyacrylamide gels to nitrocellulose sheets of a broad size range of proteins (Mr 8,000 to Mr greater than 400,000). The important features of this procedure include a two-step electrotransfer, beginning with elution of low-molecular-weight polypeptides at a low current density (approximately 1 mA/cm2) for 1 h, followed by prolonged electrotransfer (16-20 h) at high current density (approximately 3.5-7.5 mA/cm2) in conditions that favor the elution of high-molecular-weight proteins. The transfer buffer includes 0.01% sodium dodecyl sulfate to enhance protein elution, and 20% methanol to improve the retention of proteins on the nitrocellulose sheet. The nitrocellulose is air-dried after transfer is complete to eliminate loss of proteins during subsequent processing. This transfer procedure works well with proteins prepared from many different cell types, and is suitable for use with all polyacrylamide gel systems tested. With little or no modification, our method should also be applicable to transfer membranes other than nitrocellulose.     

Iodo-Gen-mediated radioiodination of nucleic acids

Iodo-Gen (1,3,4,6-tetrachloro-3a,6a-diphenylglycoluril), widely used as an oxidizing agent for iodination of proteins, can also be used for iodination of nucleic acids. Optimal conditions were determined for efficient labeling of RNA and DNA with 125I. The proposed procedure for radioiodination of nucleic acids is more beneficial than the methods utilizing TlCl3 because of the milder reaction conditions, the simplicity and completeness of separation of reaction products from the oxidizing agents, and the absence of a toxic catalyst. Using the standard procedure for Iodo-Gen-mediated iodination a specific radioactivity of up to 1.3 X 10(9) dpm/micrograms RNA can be achieved. The proposed procedure is also suitable for radioiodination of DNA.     

Preparative elution of proteins from nitrocellulose membranes after separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis

Various conditions were analyzed and optimized for the preparative elution of proteins from nitrocellulose membranes after transfer from sodium dodecyl sulfate (SDS)-polyacrylamide gels. The efficiency of elution was best using pyridine or acetonitrile elution solvents, intermediate for buffer containing a mixture of sodium dodecyl sulfate, Triton X-100, and sodium deoxycholate, and negligible for buffers containing any single detergent or chaotropic salt, such as urea or guanidine hydrochloride. The efficiency of elution with any solvent also depended on the molecular weight of the proteins, smaller proteins being more easily removed from membranes. As a general procedure, proteins may be eluted from nitrocellulose membranes by incubation with either 40% acetonitrile or 50% pyridine in 0.1 M ammonium acetate, pH 8.9, for 1-3 h at 5-37 degrees C. The recommended procedures for protein elution appear to offer a rapid, simple, and efficient means of recovering proteins from complex mixtures after separation by SDS-PAGE and transfer to nitrocellulose membranes.     

A silver-staining technique for detecting minute quantities of proteins on nitrocellulose paper: retention of antigenicity of stained proteins

A method using a silver-staining procedure to detect minute quantities of proteins on nitrocellulose paper is described. The technique is sensitive enough to detect nanogram quantities of proteins resolved on sodium dodecyl sulfate-polyacrylamide gels and then transferred to nitrocellulose paper by the electrotransfer technique. After the staining procedures, the proteins are shown to retain their antigenic properties.     

Binding of antibodies and other proteins to nitrocellulose in acidic, basic, and chaotropic buffers.

This report compares the binding of proteins to nitrocellulose membranes in acidic buffers (pH 2 and 3) with binding in neutral buffer (pH 7), basic buffers (pH 12 and 13), 8 M urea (pH 2, 3, and 7), and 6 M guanidine hydrochloride (pH unadjusted). Initially, similar amounts of antibodies and other proteins bound to the nitrocellulose membrane in all of these buffers and solvents. However, the susceptibility of individual proteins to displacement (stripping) from the membrane by the milk blocking agent depended on both the pH and the type of buffer or solvent used to bind the proteins to the membrane. Most proteins that were bound to nitrocellulose in acidic buffers were relatively resistant to milk stripping compared to proteins bound in pH 7 buffer. After correction for the amount of antibody remaining on the membrane after the milk block, it was found that acid-bound antibodies were unchanged in biological activity when compared with the same antibodies bound at neutral pH. These results suggest that acid binding of proteins could increase the sensitivity of nitrocellulose membrane assays using a milk block.     

A method for the efficient blotting of strongly basic proteins from sodium dodecyl sulfate-polyacrylamide gels to nitrocellulose

An improved procedure for the electrophoretic transfer of strongly basic proteins from sodium dodecyl sulfate-polyacrylamide gels to nitrocellulose is described. The use of more alkaline transfer buffers and the omission of an equilibration step before the transfer allow for the almost complete transfer of strongly basic proteins from gels to nitrocellulose without lowering the transfer efficiency for other proteins.