Monoclonal antibodies, electrophoretically transferred from polyacrylamide gels, retain their ability to bind specific antigens

Antibodies subjected to SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose paper, have been found to retain the ability to bind specific antigen. This has been demonstrated for two groups of antibodies, directed to a) a large molecular weight glycoprotein of the human milk fat globule and b) human interferon alpha 2. Immunoreactive antibody fragments produced by protease digestion could also be identified in this way on Western blots, thus permitting the development of optimal conditions for digestion, without the need for extensive purification procedures.     

ras p21 and other Gn proteins are detected in mammalian cell lines by [gamma-35S]GTP gamma S binding

The presence of guanine nucleotide binding proteins in mouse and human cell lines was investigated using [gamma-35S]GTP gamma S and [gamma-32P]GTP. Cell lysate polypeptides were separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis and transferred to nitrocellulose. Incubation of the nitrocellulose blots with [gamma-35S]GTP gamma S identified 9 distinct GTP-binding polypeptides in all lysates. One of these is the ras oncogene product, p21, as demonstrated by subsequent immunochemical staining of the nitrocellulose blots. We have shown that this procedure provides a sensitive method for detection of p21 in culture cell lines.     

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.     

Electronic Western blot of matrix-assisted laser desorption/ionization mass spectrometric-identified polypeptides from parallel processed gel-separated proteins

Identification of proteins previously separated by one-dimensional (1-D) or two-dimensional gel electrophoresis requires significant manipulations to digest the proteins into their respective peptides and to extract them from the gel prior to mass analysis. This article describes the simultaneous transfer and digestion of proteins directly from 1-D gels onto a membrane ready for matrix-assisted laser desorption/ionization (MALDI) mass spectrometric (MS) analysis. Protein transfer and digestion efficiencies are estimated to be more than 95%. The effectiveness of this procedure is demonstrated by identifying 110 unique proteins derived from a lysate of Escherichia coli and 149 proteins derived from a mouse liver homogenate separated by 1-D sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Using crude mouse liver homogenates, four distinct glutathione S-transferase classes, ranging from 23 to 27 kDa, are identified from a separating gel, indicating the discriminating potential for this method. A Visual Basic program allowed visualization of the identified proteins according to their respective positions on the 1-D gels. In many cases, two or more proteins could be identified within a single band of the SDS gel. The “digital” images generated resemble Western blots without the use of antibodies or signal amplification techniques.     

An electroblotting apparatus for multiple replica technique and identification of human serum proteins on micro two-dimensional gels

A new apparatus for electrophoretic transfer of proteins from micro polyacrylamide slab gels has been developed. The apparatus enabled the easy changing of nitrocellulose sheets and was suited for obtaining multiple blots from a gel. Electrophoretic conditions were determined so that all of the blots obtained sequentially from one slab gel were successfully used to visualize specific proteins irrespective of their molecular weight. Combining the transfer technique with the technique of parallel micro two-dimensional electrophoresis, 20 blots could be obtained within 1 h of electroblotting time. The locations of 28 human serum proteins were determined simultaneously on these blots using commercial specific antisera.     

"Golden blot"--detection of polyclonal and monoclonal antibodies bound to antigens on nitrocellulose by protein A-gold complexes

Protein A bound to particles of colloidal gold is widely used for cytochemical localization of antigens. A method for detecting immune complexes on "Western blots" with protein A-gold has been developed. It involved the following steps: (i) electrophoretic separation of proteins, (ii) transfer to nitrocellulose, (iii) incubation with antibodies, and (iv) staining with protein A-gold. The amount of antigen detectable was 10-50 ng. The sensitivity could be improved by subsequent photochemical silver staining (1-10 ng detected), and was then comparable to autoradiograms using iodinated protein A.     

The erasable Western blot

A method for successfully removing primary and secondary antibodies from nitrocellulose blots while preserving the originally immobilized polypeptides was developed. Polypeptides were separated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and electrophoretically transferred to nitrocellulose. Nonspecific binding sites were blocked with 5% (w/v) nonfat dried milk. After blots were reacted sequentially with antibodies directed against the antigen of interest and with radiolabeled secondary antibody, a 10-min wash in 5% (w/v) milk was required prior to drying and autoradiography. A 30-min incubation at 70 degrees C in 2% (w/v) sodium dodecyl sulfate containing 100 mM beta-mercaptoethanol quantitatively removed the antibodies and allowed reuse of the blot. A modification of this method similarly allowed reuse of Western blots when proteins were immobilized on nylon. Potential applications and limitations of this method are discussed.     

Direct tissue isoelectric focusing on ultrathin polyacrylamide gels. Applications in enzyme, lectin and immunohistochemistry

Summary Application of cryostal sections directly onto ultrathin polyacrylamide gels and subsequent isoelectric focusing allows elution of proteins, glycoproteins and peptides out of the sections into the gels. The eluted compounds reveal clearly delineated band patterns in the polyacrylamide gels. The advantage of this method is that enzyme histochemical reactions can be directly performed in the gel and in the electroeluted tissue sections. Therefore, this method is suitable for specifying, in more detail, histochemical enzyme reactions and for detecting multiple forms of enzymes even from a single tissue section. Furthermore, the transfer of proteins, glycoproteins and peptides from the gel onto nitrocellulose by a modified Western blot procedure offers the possibility of checking findings obtained by lectin histochemistry and immunohistochemistry.     

Preparation of GDP-D-mannose specifically labeled with tritium in the D-mannosyl moiety

A method, called “bidirectional transfer”, has been described for the transfer of DNA and RNA from agarose or polyacrylamide gels onto diazobenzyloxymethyl (DBM)-paper or nitrocellulose filters. The gels were sandwiched between either two nitrocellulose filters or two diazobenzyloxymethyl-papers. Next, the nucleic acids were allowed to diffuse out of the gels onto the filters. In this way, duplicate blots were obtained from a single gel. The bidirectional transfer of DNA or RNA from 0.5 to 1% agarose gels was complete and nearly quantitative after 1 h of transfer. DNA fragments from 5% polyacrylamide gels were efficiently blotted after 36 h onto nitrocellulose filters using bidirectional transfer. The fragments were transferred with good resolution and were shown to be efficient substrates for homologous [³²P]DNA probes.     

The myelin-associated glycoprotein is phosphorylated in the peripheral nervous system.

Phosphorylation of the myelin-associated glycoprotein (MAG) in the peripheral nervous system is demonstrated by immunoprecipitation from myelin proteins radiolabeled in vivo, in nerve slices and in a cell-free system. Phosphoamino acid analysis of immunoprecipitated MAG revealed the presence of radioactivity in phosphoserine, but not in phosphothreonine or phosphotyrosine. Only the shorter isoform of MAG (S-MAG) was detected by immunostaining of nitrocellulose sheets with anti-MAG anti-serum after enzymatic deglycosylation of immunoprecipitated MAG labeled in nerve slices. Autoradiography of the same Western blots revealed that most of the radioactive phosphate was in S-MAG, demonstrating that the polypeptide backbone of S-MAG is phosphorylated in the PNS.     

General detection of proteins after electroblotting by trinitrobenzene sulphonic acid derivatization and immunochemical staining with a monoclonal antibody against the trinitrophenyl group

A sensitive method for the general detection of proteins electroblotted onto nitrocellulose sheets after separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis is described. The proteins on the blots were reacted with 2,4,6-trinitrobenzene sulphonic acid. The resulting trinitrophenyl groups on the proteins were rendered visible by immunochemical staining with a monoclonal anti-trinitrophenyl antibody, and a peroxidase-conjugated second antibody. Using various proteins, the method was compared to the amidoblack method for staining of protein blots. The method was 10-100-fold more sensitive than the amidoblack method. Amounts as low as 1 ng of human serum albumin could be detected.     

Presence of guanine nucleotide-binding proteins in a plant hypocotyl microsomal fraction

The presence of specific guanine nucleotide-binding proteins in a zucchini (Cucurbita pepo L.) hypocotyl microsomal fraction was investigated. Polypeptides were separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis and transferred to nitrocellulose. Incubation of nitrocellulose blots with [alpha-32P]GTP and [gamma-32P]GTP indicated the presence of four specific and distinct GTP-binding proteins with molecular masses of approx. 23.4 kDa, 24.8 kDa, 26.6 kDa and 28.5 kDa. Binding of [alpha-32P]GTP could be completely prevented by 30 microM GDP or 10 microM guanosine 5'[gamma-thio]triphosphate. This report presents evidence for the presence in a microsomal fraction from zucchini hypocotyls of Gn-proteins as defined by Bhullar and Haslam (1987) Biochem.J. 245, 617-620. The four plant proteins resemble animal Gn-proteins when molecular weights and GTP-binding specificities are considered.     

Low molecular mass GTP-binding proteins of adrenal chromaffin cells are present on the secretory granule

Adrenal medullary homogenates and chromaffin granule membranes were separated by SDS-polyacrylamide gel electrophoresis and GTP-binding proteins detected using [alpha-32P]GTP binding to nitrocellulose blots. Four GTP-binding polypeptides of 24, 22, 20 and 18 kDa were routinely found in medullary homogenates and all were also found in isolated chromaffin granule membranes. The GTP-binding polypeptides co-sedimented with granule membrane markers following separation on sucrose gradients. On the basis of trypsin sensitivity and resistance to extraction, the GTP-binding proteins appeared to be tightly bound to the cytoplasmic surface of the granules. One or more of the secretory granule GTP-binding proteins could be involved in exocytosis in adrenal chromaffin cells.     

Studies on interaction of 5 S RNA with ribosomal proteins

Proteins of the large ribosomal subunit of rat liver (TP 60) were immobilized by diffusion transfer onto nitrocellulose after two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). Incubation of the TP 60 blots with 32P-labeled 5 S RNA under defined ionic conditions (300 mM KCl, 20 mM MgCl2) resulted in specific binding to a limited set of ribosomal proteins consisting of proteins L3, L4, L6, L13/15 and--to a lesser extent--L7 and L19. Under identical conditions, blots with proteins of the small ribosomal subunit (TP 40) did not bind 5 S RNA.     

The NADP-dependent trifunctional methylenetetrahydrofolate dehydrogenase purified from mouse liver is immunologically distinct from the mouse NAD-dependent [corrected] bifunctional enzyme

Methylenetetrahydrofolate dehydrogenase - methenyltetrahydrofolate cyclohydrolase - formyltetrahydrofolate synthetase was purified to homogeneity from mouse liver, taking advantage of its very high affinity for 2',5'-ADP-Sepharose. Antibodies raised to this trifunctional enzyme and to the bifunctional NAD-dependent dehydrogenase-cyclohydrolase from mouse Ehrlich ascites tumour cells were found not to cross-react with the purified proteins on Western blots. Each of these polyclonal antibodies detects the appropriate protein in extracts of Ehrlich ascites tumour cells after sodium dodecyl sulfate - polyacrylamide gel electrophoresis and electrophoretic transfer of the proteins to nitrocellulose. The procedure has also been used to obtain a purified preparation of the trifunctional enzyme from human liver obtained at autopsy.     

Saccharomyces cerevisiae chromatin associated protein Yp20 is a guanine nucleotide binding protein

Yp20 is a 20kD protein whose role is still obscure which copurifies with yeast histones. Yeast histones were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to nitrocellulose. Incubation of the nitrocellulose blots with [gamma-35S] GTP gamma S demonstrated that Yp20 is a GTP binding protein. A polyclonal antiserum raised against purified Yp20 cross reacted with bacterially expressed cHras and T24 Hras genuine ras products. The results obtained suggest that Yp20 is a yeast chromatin associated ras-related antigen.     

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.     

Tube-gel digestion: a novel proteomic approach for high throughput analysis of membrane proteins

This study describes a new protein digestion protocol in which a variety of detergents can be used to solubilize membrane proteins and facilitate trypsin digestion with higher efficiency. In this protocol, proteins are dissolved in solutions containing various detergents and directly incorporated into a polyacrylamide gel matrix without electrophoresis. Detergents are subsequently eliminated from the gel matrix while proteins are still immobilized in the gel matrix. After in-gel digestion of proteins, LC-MS/MS is used to analyze the extracted peptides for protein identification. The uniqueness of the protocol is that it allows usage of a variety of detergents in the starting solution without interfering with LC-MS/MS analysis. We hereby demonstrate that different detergents, including ionic SDS, non-ionic Triton X-100 and n-octyl beta-d-glucopyranoside, and zwitterionic CHAPS, can be used to achieve maximum solubilization of membrane proteins with minimal interference with LC-MS/MS analysis. Enhanced digestions, i.e. improved number and intensity of detected peptides, are also demonstrated for digestion-resistant proteins such as myoglobin, ubiquitin, and bacteriorhodopsin. An additional advantage of the Tube-Gel digestion protocol is that, even without electrophoresis separation, it allows high throughput analysis of complex protein mixtures when coupled with LC-MS/MS. The protocol was used to analyze a complex membrane protein mixture prepared from prostate cancer cells. The protocol involves only a single digestion and 2.5 h of LC-MS/MS analysis and identified 178 membrane proteins. In comparison, the same membrane fraction was resolved by SDS-PAGE, and 20 gel slices were excised and individually digested and analyzed by LC-MS/MS. The more elaborate effort demanded more than 50 h of LC-MS/MS analysis and identified 268 proteins. The new Tube-Gel digestion protocol is an alternative method for high throughput analysis of membrane proteins.     

Immunocytolocalization of glutamine synthetase in mesophyll and phloem of leaves ofSolanum tuberosum L.

Summary Localization of glutamine synthetase inSolanum tuberosum leaves was investigated by techniques of Western tissue printing and immunogold electron microscopy. Anti-GS antibodies used in immunolocalization recognize two peptides (45 kDa and 42 kDa) on Western blots. Antibody stained tissue prints on nitrocellulose membranes allowed low resolution localization of GS. Immunostaining was most evident in the adaxial phloem of the leaf midribs and petiole veins. High-resolution localization of glutamine synthetase by immunogold electron microscopy revealed that this enzyme occurs in both the chloroplasts and the cytosol ofS. tuberosum leaf cells. However, GS was specifically associated with the chloroplasts of mesophyll cells and with the cytoplasm of phloem companion cells. The evidence for cell-specific localization of chloroplast and cytosolic GS presented here agrees with the recently reported cell-specific pattern of expression of GUS reporter gene, directed by promoters for chloroplast and cytosolic GS form in tobacco transgenic plants. These data provide additional clues to the interpretation of the functional role of these different isoenzymes and its relationship with their specific localization.Abbreviations BSA bovine serum albumin - EM electron microscope - GOGAT glutamate synthase - GS glutamine synthetase - GUS -glucuronidase - IgG immunoglobulin - PBS phosphate buffer saline - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis     

DNA-binding domains of human fibronectin

Here we report the use of the technique of transferring proteins from polyacrylamide gels to nitrocellulose sheets to identify the DNA-binding domains of human plasma fibronectin. After separation of fibronectin and its tryptic digest on a sodium dodecylsulfate-polyacrylamide gel, the polypeptides were transferred to nitrocellulose sheets and the DNA-binding polypeptides were identified by incubation of the nitrocellul$\text{o}$ se sheets with ³²P-labeled human DNA followed by autoradiography.The following results were obtained: a) only two fibronectin trypsin resistant peptides (50 and 60 kd) showed DNA-binding capacity; b) in competition experiments a 300 fold excess of E. coli DNA did not inhibit the human DNA-human fibronectin binding, indicating the specificity of the interaction.