A simplified assay for cyclic AMP using protein kinase binding

Summary The protein kinase binding assay for cAMP was modified by substitution of adsorption by QAE cellulose for the membrane filtration. This modification obviates the variation of recovery of cAMP with the volume of buffer used to wash the filter. The assay is reproducible and technically simpler than those currently employed.Acknowledgment. This work was partially supported by grants HL-16583 and HL-18827 from the National Heart and Lung Institute.     

A sensitive and inexpensive high-performance liquid chromatographic assay for tyrosine hydroxylase

Summary We describe a highly sensitive assay method for tyrosine hydroxylase (TH) using high-performance liquid chromatography with amperometric determination. This assay method could be applicable to any tissues with low enzyme activity, such as rat cerebellum. We also describe the kinetic properties of TH in rat cerebral cortex.     

The use of microtiter plates for the simple and sensitive determination of insulin by an ELISA method

Summary A method of insulin determination using a commercially available ELISA kit was modified for use in microtiter plates. The adapted assay, based on the binding of procine anti-guinea pig insulin antibodies to microtiter plates and insulin-peroxidase conjugate as displacer, is sensitive between 0.5 and 30 ng/ml. Since it uses only 10–40 l of sample material it enables the determination of 5–100 pg of insulin. The rapid (5–6 h), automatable, reproducible and reliable assay makes it possible to determine many samples in a short time.     

The use of microtiter plates for the simple and sensitive determination of insulin by an ELISA method

A method of insulin determination using a commercially available ELISA kit was modified for use in microtiter plates. The adapted assay, based on the binding of porcine anti-guinea pig insulin antibodies to microtiter plates and insulin-peroxidase conjugate as displacer, is sensitive between 0.5 and 30 ng/ml. Since it uses only 10-40 microliter of sample material it enables the determination of 5-100 pg of insulin. The rapid (5-6 h), automatable, reproducible and reliable assay makes it possible to determine many samples in a short time.     

A simple method of purifying human sera for the assay of long-acting thyroid stimulator

Résumé Pour purifier les sérums pour l'étalonnage du LATS, on les agite deux fois avec l'échangeur DEAE-Sephadex. Le surnageant contient la quantité entière du LATS (96 ± 8%) et un taux fort réduit des autres protéines. Le traitement élimine la toxicité des sérums et la plupart de l'iodure et de la thyroxine sérique.

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Aided by grant No. A-4455 from the U.S. Public Health Service.     

A rapid assay for the localization of protein kinase modulator (inhibitor) in complete polyacrylamide gels

Summary A gel assay system for determination of inhibitory proteins for protein kinase(s) on isoelectric focusing gels has been developed. Preparations of heat-stable inhibitors were applied, focused, and the complete gels incubated with protein kinase in the presence of substrate protein. At the position where inhibitory protein had focused, the phosphorylation reaction was blocked selectively.This investigation was supported by the Deutsche Forschungsgemeinschaft.     

A simplified method for reading hemagglutinations on a flat-bottom microtitration plate in the mouse H-2 assay

Summary Kaliss' method for determining hemagglutination titer of mouse alloantiserum was modified to a simpler form. It became possible to read the hemagglutination directly on a flat-bottom microtitration plate using an inverted type microscope. Sensitivity of the modified method was almost the same as that of the original one. This method seems to be useful especially for the larger scale assay of H-2 specificities.Contribution from the National Institute of Genetics. Supported in part by grants-in-aid (No. 111510, 111504, 112105) from the Ministry of Education, Culture and Science, Japan.Acknowledgments. The excellent technical assistance provided by Miss M. Kuranou, Mr K. Sakakibara and Miss M. Tsuyuki is gratefully acknowledged.     

A rapid micro radial electrophoretic method of protein separation on cellulose acetate membranes.

A new rapid micromethod for protein separation under a radial electric field is described. As many as 12 rabbit serum samples could be separated in 4--6 min.     

A method for chromosome preparations from large fish specimens using in vitro short-term treatment with colchicine

This paper describes a new technique for preparing mitotic fish chromosomes using short-term in vitro treatment with colchicine. The results show that a large number of good quality metaphases (many suitable for chromosome banding) can be obtained by this technique, which requires an average of 1 h and 30 min for all steps. The procedure considerably reduces the time normally required for chromosome preparations in fish.This work was supported by grants FUNCUNESP, FAPESP and CNPq.The authors are grateful to Mr Renato Devidé for technical assistance.     

Diversity in enoyl-acyl carrier protein reductases

The enoyl-acyl carrier protein reductase (ENR) is the last enzyme in the fatty acid elongation cycle. Unlike most enzymes in this essential pathway, ENR displays an unusual diversity among organisms. The growing interest in ENRs is mainly due to the fact that a variety of both synthetic and natural antibacterial compounds are shown to specifically target their activity. The primary anti-tuberculosis drug, isoniazid, and the broadly used antibacterial compound, triclosan, both target this enzyme. In this review, we discuss the diversity of ENRs, and their inhibitors in the light of current research progress. Received 3 November 2008; received after revision 5 December 2008; accepted 8 December 2008     

Enzyme immunometric assay for the determination of pregnancy associated plasma protein A (PAPP-A) with the antigen as solid phase (conjoint IEMA)

Summary Purified pregnancy associated plasma protein A (PAPP-A) can be effectively bound to polystyrene microtitre plates. This immobilized antigen competes with the added serum PAPP-A of unknown concentration for the limited amount of peroxidase-labeled monospecific anti-PAPP-A antibody incubated simultaneously. The sensitivity is 0.2 WHO unit/ml and non-specific binding is 1.0%.Acknowledgments. My thanks go to the Janggen-Pöhn-Stiftung, St. Gallen, Switzerland, for the reception of a fellowship grant which enabled this work to be carried out. I would also like to thank Arnold Klopper, Professor of Reproductive Endocrinology, for many stimulating discussions, Garry Luke for excellent technical assistance, and the Department of Medical Illustration of the University of Aberdeen for the preparation of the graphics. The donkey serum was generously supplied by the Scottish Antibody Production Unit, Carluke, Lanarkshire.     

Enzyme immunometric assay for the determination of pregnancy associated plasma protein A (PAPP-A) with the antigen as solid phase (conjoint IEMA)

Purified pregnancy associated plasma protein A (PAPP-A) can be effectively bound to polystyrene microtitre plates. This immobilized antigen competes with the added serum PAPP-A of unknown concentration for the limited amount of peroxidase-labeled monospecific anti-PAPP-A antibody incubated simultaneously. The sensitivity is 0.1 WHO unit/ml and non-specific binding is 1.0%.