A method has been devised for the electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets. The method results in quantitative transfer of ribosomal proteins from gels containing urea. For sodium dodecyl sulfate gels, the original band pattern was obtained with no loss of resolution, but the transfer was not quantitative. The method allows detection of proteins by autoradiography and is simpler than conventional procedures. The immobilized proteins were detectable by immunological procedures. All additional binding capacity on the nitrocellulose was blocked with excess protein; then a specific antibody was bound and, finally, a second antibody directed against the first antibody. The second antibody was either radioactively labeled or conjugated to fluorescein or to peroxidase. The specific protein was then detected by either autoradiography, under UV light, or by the peroxidase reaction product, respectively. In the latter case, as little as 100 pg of protein was clearly detectable. It is anticipated that the procedure will be applicable to analysis of a wide variety of proteins with specific reactions or ligands.

https://www.pnas.org/content/pnas/76/9/4350.full.pdf

Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications.

A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity

A technique for conveniently radiolabeling DNA restriction endonuclease fragments to high specific activity is described. DNA fragments are purified from agarose gels directly by ethanol precipitation and are then denatured and labeled with the large fragment of DNA polymerase I, using random oligonucleotides as primers. Over 70% of the precursor triphosphate is routinely incorporated into complementary DNA, and specific activities of over 10(9) dpm/microgram of DNA can be obtained using relatively small amounts of precursor. These "oligolabeled" DNA fragments serve as efficient probes in filter hybridization experiments.

Labeling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase I

“Western Blotting”: Electrophoretic transfer of proteins from sodium dodecyl sulfate-polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A

http://www.alliot.fr/BIO/PDF/Southern_1975.pdf

Detection of specific sequences among DNA fragments separated by gel electrophoresis.

The structural features of nucleic acid probes tethered to a solid support and the molecular basis of their interaction with targets in solution have direct implications for the hybridization process. We discuss how arrays of oligonucleotides provide powerful tools to study the molecular basis of these interactions on a scale which is impossible using conventional analysis.

Molecular interactions on microarrays

This invention provides an apparatus and method for analyzing a polynucleotide sequence; either an unknown sequence or a known sequence. A support, e.g. a glass plate, carries an array of the whole or a chosen part of a complete set of oligonucleotides which are capable of taking part in hybridization reactions. The array may comprise one or more pair of oligonucleotides of chosen lengths. The polynucleotide sequence, or fragments thereof, are labelled and applied to the array under hybridizing conditions. Applications include analyses of known point mutations, genomic fingerprinting, linkage analysis, characterization of mRNAs, mRNA populations, and sequence determination.

Apparatus and method for analyzing polynucleotide sequences and method of generating oligonucleotide arrays

Analyzing and comparing nucleic acid sequences by hybridization to arrays of oligonucleotides: evaluation using experimental models.

A novel linker for the synthesis of oligonucleotides on a glass support is described. Oligonucleotides synthesised on the support remain tethered to the support after ammonia treatment and are shown to take part in sequence specific hybridisation reactions. These hybridizations were carried out with oligonucleotides synthesised on 'ballotini' solid sphere glass beads and microscope slides. The linker has a hexaethylene glycol spacer, bound to the glass via a glycidoxypropyl silane, terminating in a primary hydroxyl group that serves as starting point for automated or manual oligonucleotide synthesis.

Oligonucleotide hybridizations on glass supports: a novel linker for oligonucleotide synthesis and hybridization properties of oligonucleotides synthesised in situ.

Edwin M. Southern

Papers

Detection of specific sequences among DNA fragments separated by gel electrophoresis.

Molecular interactions on microarrays

Apparatus and method for analyzing polynucleotide sequences and method of generating oligonucleotide arrays

Analyzing and comparing nucleic acid sequences by hybridization to arrays of oligonucleotides: evaluation using experimental models.

Oligonucleotide hybridizations on glass supports: a novel linker for oligonucleotide synthesis and hybridization properties of oligonucleotides synthesised in situ.