Protein Blotting: A Manual
Methods of biochemical analysisPublished 16 March 1988
Jonathan M. Gershoni
Citations95
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Abstract
This chapter contains sections titled: Scope Historical Perspective A Typical Blot Experiment To Blot or Not to Blot … That Is the Question Gels The Immobilizing Matrix Protein Transfer Staining of Blots Quenching Probing and Washing Signal Detection Correlating a Signal to a Band Applications Suggested Protocols Troubleshooting Concluding Remarks
Keywords
ChemistryBiochemistry, Genetics and Molecular Biology
NatureCleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4
251,640 Citations1970Ulrich K. Laemmli
Using an improved method of gel electrophoresis, many hitherto unknown proteins have been found in bacteriophage T4 and some of these have been identified with specific gene products.
Proceedings of the National Academy of SciencesElectrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications.
53,843 Citations1979Harry Towbin, T. Staehelin +1 more
A method has been devised for the electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets that results in quantitative transfer of ribosomal proteins from gels containing urea.
Journal of Molecular BiologyDetection of specific sequences among DNA fragments separated by gel electrophoresis
32,820 Citations1975E.M. Southern
This paper describes a method of transferring fragments of DNA from agarose gels to cellulose nitrate filters that can be hybridized to radioactive RNA and hybrids detected by radioautography or fluorography.
Journal of Biological ChemistryHigh resolution two-dimensional electrophoresis of proteins.
19,340 Citations1975Patrick H. O’Farrell
This technique provides a method for estimation of the number of proteins made by any biological system and can resolve proteins differing in a single charge and consequently can be used in the analysis of in vivo modifications resulting in a change in charge.
European Journal of BiochemistryA Film Detection Method for Tritium‐Labelled Proteins and Nucleic Acids in Polyacrylamide Gels
8,651 Citations1974William M. Bonner, Ronald A. Laskey
A simple method for detecting 3H in polyacrylamide gels by scintillation autography (fluorography) using X-ray film, which is ten times more sensitive than conventional autoradiography of isotopes with higher emission energies.
Analytical Biochemistry“Western Blotting”: Electrophoretic transfer of proteins from sodium dodecyl sulfate-polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A
8,475 Citations1981W. Neal Burnette
The detection of murine leukemia virus antigens in complex cellular lysates was used to demonstrate the efficacy of this electrophoretic transfer technique.
Journal of Biochemical and Biophysical MethodsElectroblotting of multiple gels: a simple apparatus without buffer tank for rapid transfer of proteins from polycrylamide to nitrocellulose
2,520 Citations1984Jan Kyhse‐Andersen
With this apparatus, 50 protein bands from a human serum protein sample were detected by immunoblotting with the retainment of the high resolution of the SDS-PAGE technique.
Analytical BiochemistryA dot-immunobinding assay for monoclonal and other antibodies
2,189 Citations1982Richard Hawkes, Evelyn Niday +1 more
A procedure for the assay of antibodies in sera based on the application of the antigen as a spot to nitrocellulose filters is described, which has the merit of being simpler in operation and more sensitive than comparable existing procedures.
Analytical BiochemistryA rapid, sensitive method for detection of alkaline phosphatase-conjugated anti-antibody on Western blots
2,150 Citations1984M. S. Blake, Kenneth H. Johnston +2 more
A rapid, sensitive method has been developed to detect antibody-antigen complexes on "Western blots" by using Tween 20 to separate and blot the antigens onto nitrocellulose.
Journal of Biological ChemistryMolecular Weight Determination of Protein-Dodecyl Sulfate Complexes by Gel Electrophoresis in a Discontinuous Buffer System
1,998 Citations1971David M. Neville
The retardation coefficient is shown both empirically and theoretically to be a uniform function of molecular weight of protein-SDS complexes over specified ranges, providing a rationale for determining molecular weight from plots of the negative logarithm of relative mobility against molecular weight.
Gene Analysis TechniquesImproved technique utilizing nonfat dry milk for analysis of proteins and nucleic acids transferred to nitrocellulose
1,686 Citations1984David A. Johnson, James W. Gautsch +2 more
The incubation cocktail, termed BLOTTO (Bovine Lacto Transfer Technique Optimizer), is superior to bovine serum albumin or gelatin for preventing nonspecific absorption in Western blot analyses and does not require the use of detergents or chaotropic agents to effect efficient reduction of background.
Analytical BiochemistryElution of proteins from sodium dodecyl sulfate-polyacrylamide gels, removal of sodium dodecyl sulfate, and renaturation of enzymatic activity: Results with sigma subunit of Escherichia coli RNA polymerase, wheat germ DNA topoisomerase, and other enzymes
1,235 Citations1980Dayle A. Hager, Richard R. Burgess
It is shown how the method can be used to determine the approximate molecular weight of the DNA topoisomerase polypeptide by sectioning a gel on which a partially pure sample has been fractionated by electrophoresis.
Proceedings of the National Academy of SciencesRapid and sensitive colorimetric method for visualizing biotin-labeled DNA probes hybridized to DNA or RNA immobilized on nitrocellulose: Bio-blots.
1,065 Citations1983Jeffry J. Leary, David J. Brigati +1 more
At high probe concentrations (250--7560 ng/ml), biotin-labeled DNA exhibits lower nonspecific binding to nitrocellulose than does radiolabeled DNA, so hybridization times required for the analysis of unique mammalian gene sequences can be decreased to 1--2 hr.
Annual Review of BiochemistrySpecific Intermediates in the Folding Reactions of Small Proteins and the Mechanism of Protein Folding
1,033 Citations1982Peter Kim, Robert L. Baldwin
Advances in protein chemistryExperimental and Theoretical Aspects of Protein Folding
971 Citations1975Christian B. Anfinsen, H. A. Scheraga
The development of conformational energy calculation procedures will enable the three-dimensional structure of a native protein to be predicted from the knowledge of its amino acid sequence and its interactions with the solvent in which it is dissolved.
Journal of Immunological MethodsImmunoblotting and dot immunobinding — Current status and outlook
967 Citations1984Harry Towbin, J. Gordon
Analytical BiochemistryProtein blotting: Principles and applications
855 Citations1983Jonathan M. Gershoni, George E. Palade
Twenty-eight years ago, Smithies demonstrated that a starch gel could serve as a molecular sieve through which zone electrophoresis of proteins could be carried out and this procedure is now one of the most widely used analytical and preparative tools in cellular and molecular biology.
Nucleic Acids ResearchThe detection of DNA-binding proteins by protein blotting
731 Citations1980Brian C. Bowen, Jay A. Steinberg +2 more
Evidence is presented that specific protein-DNA interactions may be detected by this technique; under appropriate conditions, the lac repressor binds only to DNA containing the lac operator.
Proceedings of the National Academy of SciencesTransfer of proteins from gels to diazobenzyloxymethyl-paper and detection with antisera: a method for studying antibody specificity and antigen structure.
730 Citations1979Jaime Renart, Jakob Reiser +1 more
A rapid and very sensitive method for detecting proteins as antigens after their separation in polyacrylamide/agarose composite gels, with or without sodium dodecyl sulfate.
NatureComparative biochemical properties of normal and activated human ras p21 protein
708 Citations1984John P. McGrath, Daniel J. Capon +2 more
Human Ha-ras1 cDNAs encoding normal and activated p21 polypeptides have been efficiently expressed in Escherichia coli and the biochemical activities associated with eachpolypeptide compared and a GTPase activity is selectively impaired by a mutation which activates its oncogenic potential.
Methods in enzymology on CD-ROM/Methods in enzymology[29] Enzyme-linked immunoelectrotransfer blot techniques (EITB) for studying the specificities of antigens and antibodies separated by gel electrophoresis
677 Citations1983Victor C. W. Tsang, José Mauro Peralta +1 more
The enzyme-linked immunoelectrotransfer blot technique (EITB) combines the high resolving power of gradient sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the high sensitivity of the enzyme- linked immunosorbent assay (ELISA) to produce an extremely powerful qualitative tool for studying antigen-antibody pairs.
Analytical BiochemistryIndia ink staining of proteins on nitrocellulose paper
600 Citations1983Kathy Hancock, Victor C. W. Tsang
This stain proved to be a useful adjunct to the enzyme-linked immunoelectrotransfer blot technique and is more sensitive than Coomassie blue, amido black, and fast green stains and is simple to use.
Methods in enzymology on CD-ROM/Methods in enzymology[15] Detection of specific RNAs or specific fragments of DNA by fractionation in gels and transfer to diazobenzyloxymethyl paper
588 Citations1979James C. Alwine, David J. Kemp +5 more
The efficiency of transfer is very high and independent of the size of a fragment as covalent linkage allows convenient multiple reuse of the transfers and also allows washings to be done repeatedly under stringent conditions, thereby helping to achieve very low backgrounds.
Journal of Immunological MethodsThe use of tween 20 as a blocking agent in the immunological detection of proteins transferred to nitrocellulose membranes
557 Citations1982Byron E. Batteiger, W J Newhall +1 more
A method that blocks unoccupied protein binding sites on NCM with the nonionic detergent Tween 20, rather than proteins, proved to be equivalent or superior to other methods evaluated in the detection of immunoreactive proteins, and permitted staining of the NCM for protein after immunological probing.
The Journal of Cell BiologyIdentification and characterization of podocalyxin--the major sialoprotein of the renal glomerular epithelial cell.
516 Citations1984Dontscho Kerjaschki, David J. Sharkey +1 more
Findings indicate that the 140-kd protein is the major sialoprotein of the glomerulus, and it is the only component of glomerular lysates with an affinity for cationic dyes and lectins identical to that defined histochemically for the epithelial polyanion in situ.
Proceedings of the National Academy of SciencesSerum spreading factor (vitronectin) is present at the cell surface and in tissues.
454 Citations1983Edward G. Hayman, Michael D. Pierschbacher +2 more
The name "vitronectin" is proposed for the various forms of this protein, on the basis of its binding to glass and its adhesive properties.
Journal of Biological ChemistryElectroblotting onto activated glass. High efficiency preparation of proteins from analytical sodium dodecyl sulfate-polyacrylamide gels for direct sequence analysis.
441 Citations1986Ruedi H. Aebersold, David B. Teplow +2 more
Electrophoretic transfer (electroblotting) of proteins or their cleavage fragments onto activated glass filter paper sheets immediately after separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis has been successfully applied to the sequencing of a variety of proteins and peptides isolated from one-dimensional and two-dimensional polyacylamide gels.
Analytical BiochemistryElectrophoretic transfer of proteins and nucleic acids from slab gels to diazobenzylozymethyl cellulose or nitrocellulose sheets
438 Citations1980Michael Bittner, Patricia Kupferer +1 more
A new electrophoretic technique for transferring proteins and nucleic acids from either polyacrylamide or agarose slab gels has been developed as an alternative to blotting, and efficient transfer of DNA to both nitrocellulose membrane and diazobenzyloxymethyl cellulose is demonstrated.
Analytical BiochemistryElectrophoretic transfer of proteins from sodium dodecyl sulfate-polyacrylamide gels to a positively charged membrane filter
424 Citations1982Jonathan M. Gershoni, George E. Palade
Zeta-bind, a positively charged nylon membrane, was tested as an immobilizing matrix for the electrophoretic transfer of proteins from sodium dodecyl sulfate-polyacrylamide gels and found that Zeta- bind has a considerably greater capacity than does nitrocellulose for protein binding.
Analytical BiochemistryIdentification of concanavalin A-binding proteins after sodium dodecyl sulfate-gel electrophoresis and protein blotting
367 Citations1982Richard Hawkes
A method is described for the detection of concanavalin A-binding proteins in complex mixtures where the proteins are separated by gel electrophoresis and then blotted onto a nitrocellulose sheet where they form an immobilized replica of the original gel.
Experimental Cell ResearchVitronectin—A major cell attachment-promoting protein in fetal bovine serum
351 Citations1985Edward G. Hayman, Michael D. Pierschbacher +2 more
Using a technique in which adhesive proteins are visualized after separation by SDS-PAGE, the presence of a second cell attachment protein is graphically confirmed and evidence that this molecule is the bovine equivalent of vitronectin is presented.
Analytical BiochemistryDetection of antigens on nitrocellulose paper immunoblots with monoclonal antibodies
325 Citations1983Angel L. De Blas, Holly Cherwinski
A very sensitive method for the detection of antigen-antibody complexes on nitrocellulose paper immunoblots is described and the improved of this method is the result of the use of the detergent Tween 20 in blocking the nonspecific binding of the antibodies to the nitrocells.
Methods in enzymology on CD-ROM/Methods in enzymology[13] Immunochemical identification of membrane proteins after sodium dodecyl sulfate—polyacrylamide gel electrophoresis
307 Citations1983Albert Haid, Mordechai Suissa
Proceedings of the National Academy of SciencesUse of a protein-blotting procedure and a specific DNA probe to identify nuclear proteins that recognize the promoter region of the transferrin receptor gene.
288 Citations1985W. Keith Miskimins, M P Roberts +2 more
The technique utilizes electrophoretic transfer of NaDodSO4/PAGE-fractionated proteins onto nitrocellulose filters to identify proteins that bind specifically to the promoter region of the transferrin receptor gene.
European Journal of BiochemistryProtein-blotting on Polybrene-coated glass-fiber sheets. A basis for acid hydrolysis and gas-phase sequencing of picomole quantities of protein previously separated on sodium dodecyl sulfate/polyacrylamide gel
287 Citations1985Joel VANDEKERCHOVE, Guy Bauw +3 more
A procedure has been developed which allows the immobilization on glass-fiber sheets coated with the polyquaternary amine, Polybrene, of proteins and protein fragments previously separated on sodium-dodecylsulfate-containing polyacrylamide gels, and which was found to be more efficient and yielded binding capacities of +/- 20 micrograms/cm2.
Proceedings of the National Academy of SciencesLithium dodecyl sulfate/polyacrylamide gel electrophoresis of thylakoid membranes at 4°C: Characterizations of two additional chlorophyll a-protein complexes
287 Citations1979Philippe Delepelaire, Nam‐Hai Chua
Journal of Biological ChemistryRenaturation of enzymes after polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate.
277 Citations1980Sanford A. Lacks, S S Springhorn
A number of enzymes, including amylases, dehydrogenases, and proteases, were shown to be renaturable after polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate.
Journal of Immunological MethodsSensitive visualization of antigen-antibody reactions in dot and blot immune overlay assays with immunogold and immunogold/silver staining
273 Citations1984M. Moeremans, G. Daneels +3 more
Journal of Biological ChemistryVisualization of lipoprotein receptors by ligand blotting.
267 Citations1983Thomas O. Daniel, Wolfgang J. Schneider +2 more
In identifying LDL receptors, the ligand blotting technique is as sensitive as immunoblotting with a monoclonal antibody against the LDL receptor; it can therefore be used to identify receptors when no anti-receptor antibodies are available.
Annual Review of BiochemistryAcquisition of Three-Dimensional Structure of Proteins
249 Citations1973D.B. Wetlaufer, Sandra S. Ristow
The structure viewed as the product of the FOLDing PROCESS and how much of the PROTEin is required for self-association and self-government are compared.
Proceedings of the National Academy of SciencesA quantitative dot-immunobinding assay for proteins using nitrocellulose membrane filters.
244 Citations1984Reinhard Jahn, W Schiebler +1 more
An immunoassay method is described for the quantitative determination of synapsin I (protein I) and of a 36,000-dalton membrane protein from rat brain synaptic vesicles that should prove useful for a wide range of proteins.
Analytical BiochemistrySensitive colloidal metal (gold or silver) staining of protein blots on nitrocellulose membranes
238 Citations1985Marc Moeremans, Guy Daneels +1 more
A sensitive staining method for protein blots on nitrocellulose membranes is described and compared with commonly used dye staining methods, which will be a useful tool for correlating the position of bands or spots of proteins detected with overlay assays with the complete electropherogram in a duplicate protein blot.
Analytical BiochemistryUse of lectins for detection of electrophoretically separated glycoproteins transferred onto nitrocellulose sheets
236 Citations1981William F. Glass, Robert C. Briggs +1 more
The localization pattern of concanavalin A and wheat germ agglutinin-binding activities in the fractions from the three cell types showed the greatest similarities between the glycoprotein contents of normal liver and Novikoff hepatoma fractions.
Analytical BiochemistryCharacterization of N-linked oligosaccharides by affinoblotting with concanavalin A-peroxidase and treatment of the blots with glycosidases
221 Citations1985Loı̈c Faye, Maarten J. Chrispeels
It is shown here that the four plant glycoproteins used as models in this study interact with Con A through high-mannose oligosaccharide side chains sensitive to alpha-mannosidase and endo H degradation.
Advances in protein chemistryFolding of Protein Fragments
214 Citations1981Donald B. Wetlaufer
Folding of proteins to native-like structure has been demonstrated with fragments of 8-galactosidase, lysozyme, serum albumin, penicillinase, and tryptophan synthetase.
Journal of Biological ChemistryCalmodulin-binding proteins of the microfilaments present in isolated brush borders and microvilli of intestinal epithelial cells.
208 Citations1980John R. Glenney, K. Weber
Immunofluorescence microscopy reveals that calmodulin is present in the microvilli prior to biochemical fractionation of intestinal cells and thus is not bound artifactually during the isolation procedure, consistent with the hypothesis that the 110,000 protein is the majorCalmodulin-binding protein of the core filament structure.
NatureSynapsin I is a spectrin-binding protein immunologically related to erythrocyte protein 4.1
203 Citations1985Anthony J. Baines, Vann Bennett
It is shown that brain 4.1 is identical to the synaptic vesicle protein, synapsin I, one of the brain's major substrates for cyclic AMP and Ca2+–calmodulin-dependent kinases15,16 and Spectrin and Synapsin are present in brain homogenates in an ∼1:1 molar ratio.
Journal of Biological ChemistryBrain spectrin. Isolation of subunits and formation of hybrids with erythrocyte spectrin subunits.
193 Citations1983Jean E. Davis, Vann Bennett
Results indicate that spectrin tetramers are formed by laterally associated alpha,beta dimers attached by head-to-head linkage of each alpha chain with a beta chain, and provide strong additional evidence that brain and mammalian erythrocyte spectrin belong to a closely related family of proteins with conserved functions in both subunits.
Journal of Immunological MethodsQuantitative electrophoretic transfer of polypeptides from SDS polyacrylamide gels to nitrocellulose sheets: A method for their re-use in immunoautoradiographic detection of antigens
190 Citations1982Paul F. Erickson, Lee N. Minier +1 more
After immunoautoradiographic detection of antigens on this nitrocellulose replica with either rabbit anti-rat cerebrum immunoglobulins or mouse monoclonal antibody to a rat synaptosomal protein, the antibody and [125I]protein A were removed from the replica by treatment with 8 M urea, 0.1 M 2-mercaptoethanol, and 5 mg/ml BSA at 60 degrees C for 1 h.
Analytical BiochemistryOn the electrotransfer of polypeptides from gels to nitrocellulose membranes
190 Citations1983William Lin, Harumi Kasamatsu
The rate of elution of polypeptides from a 15% sodium dodecyl sulfate-polyacrylamide gel is dependent on the molecular weight of the individual polypepton, which is in agreement with the results of W. Burnette (Anal.
Journal of Biological ChemistryA sensitive immunoblotting method for measuring protein synthesis initiation factor levels in lysates of Escherichia coli.
184 Citations1981Jörg Howe, John W.B. Hershey
Protein synthesis initiation factor levels are measured in crude cell lysates of Escherichia coli MRE600 by use of a sensitive immunoblotting method, which serves as an alternative to the radioimmune assay in measuring small quantities of protein in complex mixtures.
Analytical BiochemistryGlycoprotein detection in nitrocellulose transfers of electrophoretically separated protein mixtures using concanavalin A and peroxidase: Application to arenavirus and flavivirus proteins
183 Citations1982J. C. S. Clegg
Glycoproteins in nitrocellulose transfers of electrophoretically separated mixtures of cellular and viral proteins are rapidly and sensitively located by sequential incubation with the lectin concanavalin A and the enzymatically active glycoprotein horseradish peroxidase.
Analytical BiochemistryProteins transferred to nitrocellulose for use as immunogens
181 Citations1985Karen A. Knudsen
Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and were eluted efficiently from the gel by transfer to nitrocellulose paper and polyclonal antibodies to the proteins were generated.
The Journal of Cell BiologyFunction of a calmodulin in postsynaptic densities. III. Calmodulin-binding proteins of the postsynaptic density.
179 Citations1981Richard K. Carlin, Dennis J. Grab +1 more
Calmodulin iodinated by the chloramine-T method, which inactivates calmodulin did not bind to the PSD but bound nonspecifically to histone and this difference between cerebral cortex and cerebellar PSDs is discussed in light of the possible function of cal modulin in synaptic excitatory responses.
The EMBO JournalThe sequence and topology of human complement component C9.
178 Citations1985Keith K. Stanley, H.P. Kocher +3 more
The amino‐terminal half of C9 is rich in cysteine residues and contains a region with a high level of homology to the LDL receptor cystein‐rich domains, and a model for C9 topology based on these findings is proposed.
The Journal of Cell BiologyMicrodomains of distinctive glycoprotein composition in the kidney proximal tubule brush border.
177 Citations1984Dontscho Kerjaschki, Lalita Noronha‐Blob +2 more
It is concluded that maltase (gp300) and the Heymann nephritis antigen (gp330) are structurally related membrane glycoproteins with a distinctive distribution in the proximal tubule brush border which may serve as markers for the microvillar and coated microdomains, respectively, of the apical plasmalemma.
Analytical BiochemistryA method for the efficient blotting of strongly basic proteins from sodium dodecyl sulfate-polyacrylamide gels to nitrocellulose
149 Citations1985Bogusław Szewczyk, Lloyd M. Kozloff
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.
The Journal of Cell BiologyCell attachment on replicas of SDS polyacrylamide gels reveals two adhesive plasma proteins.
147 Citations1982E G Hayman, Eva Engvall +4 more
This protein and fibronectin are the major attachment proteins for fibroblastic cells in plasma or serum, as defined by this assay.
Proceedings of the National Academy of SciencesDetermination of the primary amino acid sequence specifying the alpha-bungarotoxin binding site on the alpha subunit of the acetylcholine receptor from Torpedo californica.
147 Citations1985Paul T. Wilson, Thomas L. Lentz +1 more
A major determinant of the bungarotoxin-binding site on the alpha subunit resides between residues 173 and 204, the presumed site of N-linked glycosylation.
Analytical BiochemistryProtein transfer from isoelectric focusing gels: The native blot
142 Citations1982Michael P. Reinhart, Daniel Malamud
The nonelectrophoretic transfer of proteins from thin (0.5 mm) isoelectric focusing gels to nitrocellulose was complete in 1 h and the use of nondenaturing transfer buffers permits proteins to be blotted in the native state.
CellHeterogeneity of the ribosomal genes in mice and men
140 Citations1977Norman Arnheim, E.M. Southern
Studies on mouse-human cell hybrids suggest that some spacer classes are present on more than one of the five human nucleolus organizers, which indicates that ribosomal DNA in man is also heterogeneous.
Proceedings of the National Academy of SciencesMapping of the alpha-bungarotoxin binding site within the alpha subunit of the acetylcholine receptor.
140 Citations1986Drorit Neumann, D Barchan +3 more
It is demonstrated that antibodies to a synthetic peptide corresponding to residues 169-181 of the alpha subunit of the acetylcholine receptor are used to demonstrate that this sequence is included within the 18-kDa toxin binding fragment previously reported.
Journal of Immunological MethodsIdentification of surface proteins on bovine leukocytes by a biotin-avidin protein blotting technique
131 Citations1985W.L. Hurley, Eve Finkelstein +1 more
A method has been developed which covalently attaches biotin to proteins on the outer surface of leukocytes which provides a highly sensitive, non-radioactive means of examining alterations of surface proteins on leukocyte under differing functional and physiological conditions.
Journal of Biological ChemistryRestoration of 125I-alpha-bungarotoxin binding activity to the alpha subunit of Torpedo acetylcholine receptor isolated by gel electrophoresis in sodium dodecyl sulfate.
129 Citations1981John G. Haggerty, Stanley C. Froehner
Results indicate that at least part of the alpha-bungarotoxin binding site resides on the alpha subunit, which is consistent with the findings of previous work on acetylcholine receptor isolation.
Proceedings of the National Academy of SciencesImmunoautoradiographic detection of proteins after electrophoretic transfer from gels to diazo-paper: analysis of adenovirus encoded proteins.
128 Citations1981Janey Symington, M. Green +1 more
A method by which complex protein mixtures are fractionated by standard one-dimensional Na-DodSO4/polyacrylamide gel electrophoresis or O'Farrell two-dimensional gel electrosis and then are efficiently and rapidly transferred electrophoretically to diazobenzyloxymethyl- or diazophenylthioether-paper and analyzed by immunoautoradiography is described.
Analytical Biochemistry“Golden blot”—Detection of polyclonal and monoclonal antibodies bound to antigens on nitrocellulose by protein A-gold complexes
127 Citations1984D Brada, Jürgen Roth
A method for detecting immune complexes on "Western blots" with protein A-gold has been developed and was comparable to autoradiograms using iodinated protein A.
Analytical BiochemistryMultiple immunoreplica technique: Screening for specific proteins with a series of different antibodies using one polyacrylamide gel
125 Citations1981Roman P. Legocki, Desh Pal S. Verma
Identical size of the replicas and superimposable profiles of proteins detected by antibodies allow a precise localization of particular polypeptides in the original gel.
Journal of Biological ChemistryBinding sites for lactogenic and somatogenic hormones from rabbit mammary gland and liver.
125 Citations1983M T Haeuptle, Michel L. Aubert +2 more
The Journal of Cell BiologyLocalization of cellular antigens in sodium dodecyl sulfate-polyacrylamide gels.
119 Citations1978W. Steven Adair, D.A. Jurivich +1 more
A procedure is described for localizing antigen-antibody complexes in sodium dodecyl sulfate (SDS) polyacrylamide gels using 125I-labeled protein A from Staphylococcus aureus to probe antigenic cross-reactivities between Strongylocentrotus and Chlamydomonas alpha- and beta-tubulins.
Proceedings of the National Academy of SciencesIsolation and biochemical characterization of the mammalian reovirus type 3 cell-surface receptor.
118 Citations1985M S Co, Glen N. Gaulton +2 more
Comparison of the reovirus receptor on murine thymoma and rat neuroblastoma cells indicated that similar structures on the cell surface are recognized by theReovirus type 3 and the anti-receptor antibodies as previously suggested from cellular and binding studies.
Biochemical and Biophysical Research CommunicationsConditions for reproducible detection of calmodulin and S100β in immunoblots
116 Citations1984Linda J. Van Eldik, Sandra R. Wolchok
An immunoblot protocol is provided that allows the rapid and reproducible detection of calmodulin and S100β in crude biological samples and is described here some of the basis of this difficulty.
Proceedings of the National Academy of SciencesBinding of alpha-bungarotoxin to isolated alpha subunit of the acetylcholine receptor of Torpedo californica: quantitative analysis with protein blots.
114 Citations1983Jonathan M. Gershoni, Edward Hawrot +1 more
It is demonstrated that isolated, immobilized alpha subunit of the Torpedo acetylcholine receptor can bind alpha-bungarotoxin, however, the observed binding of alpha- bungarOToxin to immobilizedalpha subunit is reduced in affinity to 1/1,000 to 1 /10,000 of that obtained with native receptor.
Journal of Immunological MethodsApplication of the nitrocellulose transfer technique and alkaline phosphatase conjugated anti-immunoglobulin for determination of the specificity of monoclonal antibodies to protein mixtures
112 Citations1982Carolyn O'Connor, Leonie K. Ashman
The target antigens of a series of monoclonal antibodies raised against a complex mixture of proteins have been identified by incubating nitrocellulose strips with the 'hybridoma' culture supernatants and detecting bound antibody using alkaline phosphatase conjugated to rabbit anti-mouse F(ab')2 antibody, and histochemical substrates.
ElectrophoresisSpecific antiserum staining of two‐dimensional electrophoretic patterns of human plasma proteins immobilized on nitrocellulose
111 Citations1982N. Leigh Anderson, N. Leigh Anderson +4 more
The transfer technique constitutes a generally useful immunological “third‐dimension” in the high‐resolution separation of proteins and seems likely that a large majority of proteins retain sufficient conformation throughout the analytical procedure (or can regain it easily afterwards) to be recognized immunologically.
FEBS LettersVacuum‐blotting: a new simple and efficient transfer of proteins from sodium dodecyl sulfate—polyacrylamide gels to nitrocellulose
109 Citations1982M. Peferoen, Roger Huybrechts +1 more
The transfer of proteins from polyacrylamide gels to nitrocellulose paper has been of great interest since a wide variety of analytical procedures can be applied to the immobilized proteins.
Journal of Biological ChemistryMonoclonal antibodies against eucaryotic ribosomes. Use to characterize a ribosomal protein not previously identified and antigenically related to the acidic phosphoproteins P1/P2.
109 Citations1982Harry Towbin, Hans-Peter Ramjoué +3 more
The common determinant on proteins P1 and P2 cross-reacted with proteins of similar molecular weight in all eucaryotes tested, and with a determinant in a previously uncharacterized 38,000-dalton protein of the large ribosomal subunit, which is designated P0.
Analytical BiochemistryProtein blotting: Detection of proteins with colloidal gold, and of glycoproteins and lectins with biotin-conjugated and enzyme probes
107 Citations1985R. Rohringer, David W. Holden
Gold sols were found to be useful as general stains for proteins: They are polychromatic, yield an indelible record, and are complementary to india ink as protein stains because these two stains have different sensitivities for a number of proteins tested.
International review of cytologyDo Diploid Fibroblasts in Culture Age?
104 Citations1979Eugene Bell, Louis F. Marek +4 more
In the intact organism, fibroblasts may be called upon to participate in wound healing and connective tissue restructuring and are considered stem cells in the sense that basal epithelial cells or bone marrow cells are stem cells, because stem cells are programmed to divide on a more or less regular basis.
Journal of Immunological MethodsUse of a zwitterionic detergent for the restoration of the antibody-binding capacity of electroblotted meningococcal outer membrane proteins
102 Citations1984Robert E. Mandrell, Wendell D. Zollinger
Meningococcal class 2 proteins which had lost their earlier capacity to bind mouse monoclonal antibodies in the normal blot procedure after SDS-polyacrylamide gel electrophoresis, bound monoconal antibodies following the addition of the zwitterionic detergent to the blot buffer.
Analytical BiochemistryImmunogold for detection of antigen on nitrocellulose paper
100 Citations1984Yau‐Heiu Hsu
This method can detect 1-5 pg of TMV protein, either in purified form or in the unpurified plant extract, with high specificity, and was detected by gold-labeled goat anti-rabbit immunoglobulin G.
Nucleic Acids ResearchSolubilization and immune-detection of β-galactosidase hybrid proteins carrying foreign antigenic determinants
97 Citations1983Keith K. Stanley
This model system demonstrates a simple procedure for identifying DNA exon fragments by the immunological detection of expressed hybrid proteins in a high percentage of colonies.
The Journal of Cell BiologyCalmodulin-stimulated protein kinase activity from rat pancreas.
94 Citations1983Fred S. Gorelick, Jonathan Cohn +4 more
Journal of Clinical InvestigationIdentification of a major sialoprotein in the glycocalyx of human visceral glomerular epithelial cells.
91 Citations1986Dontscho Kerjaschki, H Poczewski +5 more
A major membrane sialoprotein in human glomeruli that is similar to rat podocalyxin in its sialic acid-dependent binding of wheat germ agglutinin and in its localization on the surface of glomerular epithelial and endothelial cells is identified.
The EMBO JournalThe 43‐K protein, v1, associated with acetylcholine receptor containing membrane fragments is an actin‐binding protein.
88 Citations1984John H. Walker, Catherine M. Boustead +1 more
The findings suggest that the 43 000 mol.
Analytical BiochemistryA silver stain for the rapid quantitative detection of proteins or nucleic acids on membranes or thin layer plates
86 Citations1986Carl R. Merril, Mary E. Pratt
This stain demonstrates a reproducible curvilinear relationship between silver density and the amount of protein or DNA, over an averaged concentration range from 1 to 300 ng for proteins and 10 to 710 ng for DNA.
Analytical BiochemistryMicroassay for proteins on nitrocellulose filter using protein dye-staining procedure
85 Citations1985Kazuyuki Nakamura, Tatehiko Tanaka +2 more
A simple and rapid microassay for proteins utilizing the protein dye-staining procedure with a nitrocellulose (NC) filter is described, with good linearity between the optical density and the amount of protein obtained.
Proceedings of the National Academy of SciencesAntibodies to synthetic peptides as probes for the binding site on the alpha subunit of the acetylcholine receptor.
84 Citations1985Drorit Neumann, Jonathan M. Gershoni +2 more
The results indicate that the toxin-binding site lies beyond the first possible V8 protease cleavage site after residues 126-143: i.e., Asp-152, in agreement with the possibility that cysteine residues 192 and/or 193 are in close proximity to or contiguous with the ligand- binding site.
Journal of Experimental ZoologyIdentification of zona binding proteins of rabbit, pig, human, and mouse spermatozoa on nitrocellulose blots
83 Citations1985Michael G. O’Rand, Jessica Matthews +2 more
The homologous zona binding proteins (ZBP) of ejaculated rabbit, pig and human spermatozoa and epididymal mouse spermatoza have been identified and the rabbit's ZBPs have relative molecular weights of 32K, 18K, 16K and 14K; the pig's major ZBP is 16K while human spermutozoa bind human zona protein at 17K and 18K.
Journal of Biological ChemistryMetabolism of prostacyclin in the rabbit kidney.
82 Citations1979P Y Wong, J C McGiff +3 more
Prostacyclin infused into the kidney and the radiolabeled products from the renal venous effluent were separated by thin layer chromatography and identified by gas chromatography-mass spectrometry.
Analytical BiochemistryPreparative elution of proteins from nitrocellulose membranes after separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis
80 Citations1985Bharat Parekh, Hina Mehta +2 more
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.
Analytical Biochemistry3-(N-maleimido-propionyl) biocytin: A versatile thiol-specific biotinylating reagent
78 Citations1985Edward A. Bayer, Mariano Zalis +1 more
A biotin-containing, thiol-specific reagent, 3-(N-maleimido-propionyl) biocytin (MPB), was synthesized and used to biotinylate various proteins via native or artificially induced sulfhydryl groups, and could be used to detect protein SH groups on dots with sensitivities in the femtomole range.
Analytical BiochemistryProtease-facilitated transfer of high-molecular-weight proteins during electrotransfer to nitrocellulose
77 Citations1981Wade Gibson
The technique takes advantage of in situ proteolysis to reduce the size of large proteins to enhance the electrophoretic transfer of high-molecular-weight proteins from polyacrylamide gels onto nitrocellulose.
Proceedings of the National Academy of SciencesBinding of alpha-bungarotoxin to proteolytic fragments of the alpha subunit of Torpedo acetylcholine receptor analyzed by protein transfer on positively charged membrane filters.
73 Citations1984Paul T. Wilson, Jonathan M. Gershoni +2 more
It is demonstrated that immobilized proteolytic fragments as small as 1/5 the size of the alpha subunit retain the structural characteristics necessary for binding alpha-bungarotoxin, although the toxin is bound to the fragments with lower affinity than to the native receptor.
Analytical BiochemistryProtein blotting in uniform or gradient electric fields
73 Citations1985Jonathan M. Gershoni, Fred E. Davis +1 more
Transfer apparatus with either uniform or gradient electric fields are expected to improve qualitative results and make possible quantitation of protein blotting.
Biochemical and Biophysical Research CommunicationsDefinition of the antigenic polypeptides in the Sm and RNP ribonucleoprotein complexes
72 Citations1981Philip J. White, Sallie O. Hoch
RNA was necessary for activity when assayed by counterimmunoelectrophoresis, as demonstrated by RNase sensitivity, suggesting a role for RNA in mediating a precipitin reaction of the two antigens.
Analytical Biochemistry125I-wheat germ agglutinin blotting: Increased sensitivity with polyvinylpyrrolidone quenching and periodate oxidation/reductive phenylamination
68 Citations1984James R. Bartles, Ann L. Hubbard
Improved labeling with 125I-wheat germ agglutinin is observed for orosomucoid derivatives that display N-acetylglucosamine or sialic acid residues at the nonreducing termini of their oligosaccharides, as well as for a number of glycoprotein components of a rat hepatocyte plasma membrane fraction.
Journal of Immunological MethodsThe use of alkaline-phosphatase-conjugated second antibody for the visualization of electrophoretically separated proteins recognized by monoclonal antibodies
67 Citations1983Bryan M. Turner
An immunoenzyme procedure is described for detection of proteins separated by SDS-polyacrylamide gel electrophoresis that can detect protein antigens at concentrations down to 0.5 ng/mm2.
Analytical BiochemistryCalmodulin-binding proteins: Visualization by 125I-calmodulin overlay on blots quenched with tween 20 or bovine serum albumin and poly(ethylene oxide)
67 Citations1984Steven D. Flanagan, Beverly Yost
To streamline detection of calmodulin-binding proteins, blotting techniques for the electrophoretic transfer of proteins onto nitrocellulose filters, followed by overlay with 125I-calmodulin, have been adapted.
Methods in enzymology on CD-ROM/Methods in enzymology[31] Selection of specific clones from colony banks by screening with radioactive antibody
66 Citations1979Louise Clarke, Ronald A. Hitzeman +1 more
The plastic well method and the agar plate–paper disk method for screening colony banks with radioactive antibody are described, which avoids selective pressure for a functional expression and the structural alterations within the cloned DNA segment resulting in an efficient expression in the bacterial cell would not be expected to occur.
Analytical BiochemistryBlot analyses of glycoconjugates: Enzyme-hydrazide—a novel reagent for the detection of aldehydes
66 Citations1985Jonathan M. Gershoni, Edward A. Bayer +1 more
A procedure for the generalStaining of glycoproteins and other glycoconjugates on protein blots has been developed and the new assay is advantageous over periodic-acid Schiff staining of gels as its reagents and signals are stable and the process is simple and expedient, and provides greater sensitivity.
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