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P L Bloch

Publications and source records attributed to P L Bloch.

11 recordsLinked to original sources
PubMed1991

Temperature cycler evaluation: what do you need to know?

Which of the 20 different, commercially available, temperature cyclers adequately fulfill all functional requirements? How can an investigator analyze instrument function? This report describes in detail, for the first time, the characteristics of Ericomp temperature cyclers. The data show that the temperature uniformity, accuracy and maximum overshoot is +/- 0.34 degree C, less than 0.5 degree C and 0.17 degree C, respectively. Cycling temperature reproducibility is 0.1%, and cycling time reproducibility is 0.4%. These data compare very favourably to published data describing other temperature cyclers. The techniques used to obtain these data serve as a model for evaluating instruments for well-to-well uniformity, temperature accuracy and within-run reproducibility.

Biotechnology↗
PubMed1987

The glycine-alanine repeating region is the major epitope of the Epstein-Barr nuclear antigen-1 (EBNA-1).

The Epstein-Barr nuclear antigen-1 (EBNA-1) is a protein containing a large glycine-alanine repeat that has been shown to be antigenic. Antibodies to EBNA-1 can be detected by means of immunoblotting. Preincubation of antisera with purified EBNA-1 protein inhibits the binding of IgG antibodies in this system, indicating that those epitopes detected by immunoblots are also accessible on the native molecule. A number of synthetic peptides the sequences of which were derived from the glycine-alanine repeating region of EBNA-1 and from regions adjacent to it also inhibited antibody binding to EBNA-1. These showed, however, a 1000-fold variation in their inhibitory activities. Peptides containing only glycine and alanine were the most effective inhibitors. The anti-EBNA-1 antibodies did not react with several other peptides representing sequences from unrelated proteins. At saturating concentrations of peptide 85 to 100% of anti-EBNA-1, antibody binding was inhibited in all sera tested with one exception. Similar results are obtained when antibody binding is assayed by an enzyme immunosorbent assay by using partially purified EBNA-1 to coat the plates. Thus the glycine-alanine region, either through its primary structure or through conformations assumed by this region, forms the major epitope(s) of the EBNA-1 molecule.

Alanine↗
PubMed1981

Multiple forms of lysyl-transfer ribonucleic acid synthetase in Escherichia coli.

Lysyl-transfer ribonucleic acid synthetase (EC 6.1.1.6) was identified as four polypeptide spots after two-dimensional polyacrylamide gel electrophoresis of whole-cell lysates of Escherichia coli. Identification was made by migration with partially purified enzyme preparations, by peptide map patterns, by mutant analysis, and by correlation of spot intensities with changes in enzyme levels under different growth conditions. Wild-type cells growing at 37 degrees C in glucose minimal medium displayed the enzyme predominantly as two spots (spots I and III). Growth at 46 degrees C, growth in the presence of alanine or glycyl-L-leucine, or growth of a strain with a mutational deficiency in S-adenosylmethionine synthetase (metK) greatly increased the synthesis of two other spots (spots II and IV). Polypeptides I and III, but not polypeptides II and IV, had altered isoelectric points in a lysyl-transfer ribonucleic acid synthetase mutant. These data suggest that multiple forms of lysyl-transfer ribonucleic acid synthetase exist in vivo and that they may be encoded by more than one gene.

Amino Acyl-tRNA Synthetases↗
PubMed1980

Protein identifications of O'Farrell two-dimensional gels: locations of 81 Escherichia coli proteins.

The two-dimensional gel electrophoresis method of P. H. O'Farrell readily resolves approximately 1,000 proteins from whole-cell homogenates. We have found that the location of most individual proteins is sufficiently reproducible and precise to permit different laboratories to exchange information about them. We present the location of 81 Escherichia coli structural proteins, binding proteins, enzymes, and factors, identified with the aid of purified proteins supplied to us by many investigators.

Bacterial Proteins↗
PubMed1980

Protein identifications on O'Farrell two-dimensional gels: locations of 55 additional Escherichia coli proteins.

The resolution of proteins from whole-cell homogenates by two-dimensional gel electrophoresis is sufficiently reproducible and precise to permit different laboratories to exchange information about them. To the previous total of 81 we add the locations of 55 Escherichia coli proteins determined with the aid of purified proteins and mutant strains supplied by many investigators. The criteria used to establish the identifications of protein spots include migration with marker proteins, altered position or amount in appropriate mutant or plasmid-carrying strains, physiological behavior, and peptide map pattern.

Bacterial Proteins↗
PubMed1978

Patterns of protein synthesis in E. coli: a catalog of the amount of 140 individual proteins at different growth rates.

The amount of 140 individual proteins of E. coli B/r was measured during balanced growth in five different media. The abundance of each protein was determined from its absolute amount in 14C-glucose-minimal medium and a measurement of its relative amount at each growth rate using a double labeling technique. Separation of the proteins was carried out by two-dimensional gel electrophoresis. This catalog of proteins, combined with 50 additional ribosomal proteins already studied, comprises about 5% of the coding capacity of the genome, but accounts for two thirds of the cell's protein mass. The behavior of most of these proteins could be described by a relatively small number of patterns. 102 of the 140 proteins exhibited nearly linear variations with growth rate. The remaining 38 proteins exhibited levels which seemed to depend more on the chemical nature of the medium than on growth rate. Proteins, including the ribosomal proteins, that increase in amount with increasing growth rate account for 20% of total cell protein by weight during growth on acetate, 32% on glucose-minimal medium and 55% on glucose-rich medium. Proteins with invariant levels in the various media comprise about 4% of the cell's total protein.

Bacterial Proteins↗
PubMed1977

Chemical measurement of steady-state levels of ten aminoacyl-transfer ribonucleic acid synthetases in Escherichia coli.

Polypeptide chains of 10 aminoacyl-transfer ribonucleic acid synthetases (those for arginine, glutamine, glutamic acid, glycine, isoleucine, leucine, lysine, phenylalanine, threonine, and valine) have been identified in lysates of Escherichia coli resolved by the O'Farrell two-dimensional gel system. By labeling cells uniformly with [14C]glucose and by measuring the total amounts of these polypeptides by their radioactivity, estimations of the steady-state, molecular amounts of these enzymes were made and compared to the number of ribosomes and elongation factors in these cells. Portions of a reference culture grown on glucose and labeled with [14C]leucine or [35S]sulfate were mixed with four cultures grown in widely different media containing [3H]leucine or [3H]leucine plus [3H]isoleucine. From the isotope ratios of the total protein and of the spots containing the synthetase chains, the chemical amount of each synthetase relative to that of the reference culture was determined. The results, where comparable, show reasonable agreement with enzyme activity measurements. In general, these synthetases each exhibit a positive correlation with growth rate in unrestricted media, indicating a strong tendency for the levels of transfer ribonucleic acid, synthetases, elongation factors, and ribosomes to remain approximately, though not exactly, in balance at different growth rates.

Alanine-tRNA Ligase↗
PubMed1974

Culture medium for enterobacteria.

A new minimal medium for enterobacteria has been developed. It supports growth of Escherichia coli and Salmonella typhimurium at rates comparable to those of any of the traditional media that have high phosphate concentrations, but each of the macronutrients (phosphate, sulfate, and nitrogen) is present at a sufficiently low level to permit isotopic labeling. Buffering capacity is provided by an organic dipolar ion, morpholinopropane sulfonate, which has a desirable pK (7.2) and no apparent inhibitory effect on growth. The medium has been developed with the objectives of (i) providing reproducibility of chemical composition, (ii) meeting the experimentally determined nutritional needs of the cell, (iii) avoiding an unnecessary excess of the major ionic species, (iv) facilitating the adjustment of the levels of individual ionic species, both for isotopic labeling and for nutritional studies, (v) supplying a complete array of micronutrients, (vi) setting a particular ion as the crop-limiting factor when the carbon and energy source is in excess, and (vii) providing maximal convenience in the manufacture and storage of the medium.

Ammonia↗