Quantitative assay of the binding of small molecules to protein: comparison of dialysis and membrane filter assays

A rapid assay involving filtration through a nitrocellulose filter is described for the quantitative detection of a protein which specifically and reversibly binds a small molecule. This assay is quantitatively characterized by direct comparison with equilibrium dialysis. The filtration assay is highly sensitive and reproducible when applied to the binding of histidine by the J protein, a component of histidine transport. The effects of several variables on this method are examined. Also, an equilibrium dialysis procedure designed for optimal sensitivity and range in the assay of proteins by binding activity is described.     

A simplified assay for the enzyme responsible for the attachment of myristic acid to the N-terminal glycine residue of proteins, myristoyl-CoA: glycylpeptide N-myristoyltransferase.

A greatly simplified assay for myristoyl-CoA:glycylpeptide N-myristoyltransferase (NMT) activity is described. The assay is based on the differential solubility of the acyl-peptides produced as a consequence of the NMT activity and yields results comparable with those obtained with the original assay described by Towler & Glaser [(1986) Proc. Natl. Acad. Sci. U.S.A. 83, 2812-2816], which requires h.p.l.c. to determine the production of the acyl-peptides. The use of the revised assay in the preliminary steps of the purification of rat brain NMT is described, and its use in determining the fatty acid-specificity of the enzyme is illustrated. The results are shown to be comparable with those obtained with the h.p.l.c.-based assay.     

Modified radiorespirometric assay for determining the sulfate reduction activity of biofilms on metal surfaces

A field method is described for the assay of [³⁵S]sulfate reduction by sulfate-reducing bacteria in biofilms on metal surfaces. The assay is such that the biofilm can be studied without removing it from the substratum. The presence of the metal coupons, however, required preliminary optimisation of conditions for accurate determination of in situ sulfate reduction rates. Modifications to the radiorespirometric assay are described and successful field trials are presented.     

Fluorometric assays for succinyl-CoA and propionyl-CoA in mitochondrial extracts

Fluorometric assay procedures are described for the quantitative measurements of succinyl-CoA and propionyl-CoA down to concentrations of 0.1 μm in the reaction mixture. The enzymatic assay for succinyl-CoA couples the reaction of 3-ketoacid CoA transferase (succinyl-CoA transferase) to β-OH butyryl-CoA dehydrogenase. A simple purification procedure is described for the isolation of succinyl-CoA transferase from beef heart. Two enzyme assays for propionyl-CoA are described. In the first, CoA, acetyl-CoA and propionyl-CoA are assayed by sequential addition of α-ketoglutarate dehydrogenase, citrate synthase and phosphotransacetylase. The second assay for propionyl-CoA utilized propionyl-CoA carboxylase to convert propionyl-CoA to methylmalonyl-CoA in the presence of ATP and bicarbonate, and the ADP formed was assayed by coupling pyruvate kinase with lactate dehydrogenase. Illustrations are given for the application of these assay procedures to measurements of succinyl-CoA and propionyl-CoA in neutralized perchloric acid extracts prepared from rat heart and liver mitochondria incubated under a variety of conditions.     

Multiplex genotyping of PCR products with MassTag-labeled primers.

A simple mass spectrometric based assay, the PinPoint assay, has previously been described for typing single nucleotide polymorphisms. The identity of a polymorphism is determined by mass differences of single base extended genotyping primers as determined by MALDI-TOF mass spectrometry. A simple method for multiplexing the assay is described, employing multiple primers with 5'oligo(dT) sequences (MassTags) which serve to mass discriminate the peaks of multiple extended and non-extended primers. The assay is extremely rapid and requires no labeling reagents.     

Spectrophotometric Characteristics and Assay of Biological Stains

I. The chemical and spectrophotometric methods adopted in the Stain Commission laboratories for the certification of stains are described. Spectrophotometric examination is by the Beckman spectrophotometer in which the color density at the absorption maximum is determined as well as a ratio of color densities designed to detect abnormal symmetry of the absorption curve. A somewhat modified titanous chloride assay is described; eerie sulfate is used to determine the normality of the unstable titanous chloride, and the oxidant solution, which is stable, is standardized against Bureau of Standards arsenious oxide. The general approach used for re-study of the stains by these methods and the criteria necessary for adoption of the spectrophotometric method of assay are described.

II. This report gives the details of the spectrophotometric examination and assay of the individual stains in the thiazin group, namely methylene blue, thionin, methylene violet, toluidine blue O, and azures A, B, and C. The spectrophotometric method of assay was adopted for the first three named; the others showed too large a variation among samples to permit adoption of this simpler type of assay. In the case of methylene violet it was necessary to do nitrogen analyses to relate purity of the dye and color density. Complete absorption curves for typical samples of each of the above stains are presented.     

Rabbit lutropin: preparation, characterization of the hormone, its subunits and radioimmunoassay.

The purification of rabbit lutropin is described. A product with a potency of 1.53 X NIH-LH-Sl was obtained as assayed by the ovarian ascorbic acid depletion assay. In a homologous radioimmunoassay, which is described, rabbit lutropin has a potency 4.83 X NIH-LH-Sl. In a radioligand assay, utilizing labeled ovine lutropin as the trace, the relative potency was 0.47 X NIH-LH-Sl measured by 50% inhibition comparison since rabbit lutropin response in this system did not parallel ovine lutropin. A counter-current distribution procedure for separation of rabbit lutropin subunits is described. Amino acid composition of the isolated subunits and intact rabbit lutropin was determined. The carbohydrate composition of the latter is presented; only amino sugar determinations are available for the subunits. The NH2-terminal amino acids are phenylalanine (alpha subunit) and alanine (beta subunit). Preliminary data on COOH-terminal amino acids are provided.     

Measurements of serum glucose using the luciferin/luciferase system and a liquid scintillation spectrometer

A single-step assay for serum glucose measurements is described. The assay is based on the phosphorylation of D-glucose by glucokinase and the measurement of ATP consumption by firefly luciferase. The luminescence is recorded in an ordinary liquid scintillation spectrometer. The use of stable reagents and a stable final signal (light emission) makes it possible to analyze a large number of samples in each assay run. The assay is of particular value when repeated serum glucose determinations are performed on samples from small laboratory animals.     

Radioimmunoassay of PGE and an approach to the specific measurement of PGE1

A specific radioimmunoassay for prostaglandins of the E series is presented. Several refinements of the conventional procedures used for unknown sample preparation prior to assay are described. Validation of the method through recovery of labeled and unlabeled prostaglandin E from plasma indicates: 74% extraction efficiency, 80% chromatographic separation efficiency, and a 67% overall recovery figure for the complete assay. The development of an “absorbed system” approach for measuring one E prostaglandin (PGE₁) without interference from the other E prostaglandin (PGE₂) is also described. A preliminary study of rat tissue and male plasma samples measured by these assay systems is included. Alternate approaches to the development of a specific PGE₁ assay, although less feasible in these authors' hands, are discussed.     

An ultrafiltration assay for lysyl oxidase

A modification of the original microdistillation assay for lysyl oxidase is described in which Amicon C-10 microconcentrators are used to separate, by ultrafiltration, the 3H-labeled products released from a [4,5-3H]-lysine-labeled elastin substrate. Enzyme activity is determined by scintillation counting of the ultrafiltrate, after subtraction of radioactivity released in the presence of beta-aminopropionitrile, a specific inhibitor of the enzyme. Conditions are described which optimize both the sensitivity and the efficient use of substrate. The assay shows linear inhibition of activity in up to 1 M urea; hence, as the enzyme is normally diluted in the assay, samples in 6 M urea can be assayed directly, without prior dialysis, and corrected for partial inhibition. Comparable results are obtained when enzyme activity is assayed by ultrafiltration or microdistillation. The assay is simple and convenient and, by using disposable containers throughout, it eliminates the need for time-consuming decontamination of radioactive glassware.     

Development of a gas chromatographic selected ion monitoring assay for thyroxine (T4) in human serum

The components of a gas chromatographic mass spectrometric-selected ion monitoring (SIM) assay for thyroxine (T4) in human serum are described. The internal standard for the assay was synthesized from deuterium-labelled 3,5-diiodotyrosine and 3,5-diiodo-4-hydroxyphenylpyruvic acid. A novel method was developed for isolating the products of the coupling reaction. The results obtained by gas chromatography mass spectrometry SIM were compared with those of radioimmunoassay. The gas chromatographic mass spectrometric SIM assay would form the basis of a reference assay for T4.     

Mutagenicity and DNA- modifying activity: A comparison of two microbial assays

Aspects of the Salmonella mutagenesis and Escherichia coli DNA polymerase deficient (pol A1^?) assay procedures for detecting environmental mutagens are discussed.The chief limitation of the pol A1^?1- assay involves substances that do not diffuse rapidly in agar. This problem can be overcome by performing the test in suspension. A simple procedure for accomplishing this is described.Although the Salmonella assay is more flexible, under routine conditions it does not respond to several classes of substances which give positive responses in the pol A1^?1 system.For optimal testing, it is recommended that the two microbial assays be used in tandem.The DNA-modifying properties of povidone-iodine for eukaryotic and prokaryotic cells are described. Even though this substance does not display mutagenic properties in the standard Salmonella assay, it does so in suspension culture. The basis of the mutagenic and DNA-modifying properties of povidone-iodine appears to involve the iodination of the cystosine residue of DNA.     

A reverse-phase liquid chromatographic assay for flavin-containing monooxygenase activity

A rapid, convenient assay for flavin-containing monooxygenase activity is described. The method is based on direct analysis of quenched incubation mixtures by reverse-phase liquid chromatography, and utilizes p-nitrophenyl-1,3-oxathiolane as the substrate. The synthesis of the substrate and the product are described. The usefulness of p-nitrophenyl-1,3-oxathiolane S-oxide formation as a measure of flavin-containing monooxygenase activity was demonstrated using highly purified and microsomal hog and rat liver flavin-containing monooxygenase. The assay is especially useful for determining stereoselectivity of flavin-containing monooxygenase activity in small amounts of crude tissue preparations.     

Problems associated with the assay of arylsulphatases A and B of rat tissues

A method for the separation and purification of rat liver arylsulphatases A and B by gel filtration on Sephadex G-200 is described. The properties of the A enzyme and its molecular weight are similar to those of the corresponding ox liver enzyme. The B enzymes were found to be dissimilar. The method already developed for the assay of the corresponding enzymes from human tissues was shown to be unsuitable for the assay of the enzymes of rat tissues. A method of assay was developed which permits an approximate determination of the individual rat liver enzymes in a mixture of the two, but precise determination requires prior separation of the enzymes by gel-filtration chromatography.     

Quantitative in situ gel electrophoretic assay for neomycin phosphotransferase activity in mammalian cell lysates

A method for the assay of neomycin phosphotransferase activity in eucaryotic cell lysates is described. Total cytoplasmic proteins are fractionated in nondenaturing polyacrylamide gels and then allowed to react in situ with [gamma-32P]ATP and kanamycin. The reaction products are detected by blotting to phosphocellulose paper and autoradiography. The assay is linear with protein concentration and sensitive enough to detect expression in transient assays.     

检测特异性抗体分泌细胞(ASCs)的固相酶联免疫斑(ELISPOT)技术的建立

ＥＬＩＳＰＯＴ技术是目前国外评价疫苗免疫原性和免疫应答机理研究中最常使用的方法。本文用自制的ＮＣ膜２４孔板为固相支持物,建立了检测小鼠脾细胞中特异性ＡＳＣｓ的ＥＬＩＳＰＯＴ技术,在检测方法的特异性、结果的稳定性上均取得了满意的效果。并对该方法的某些实验条件进行了摸索。     

A fluorometric assay for the potassium-dependent phosphatase activity of the (Na+ + K+)-adenosine triphosphatase

A fluorometric assay for the K+-dependent phosphatase activity of the (Na+ + K+)-ATPase in both purified and membrane-bound forms is described. The assay utilizes 3-O-methylfluorescein phosphate as substrate and measures the fluorescence of the 3-O-methylfluorescein produced by hydrolysis of the substrate. The assay described is an order of magnitude more sensitive than the assay employing p-nitrophenylphosphate, the substrate most commonly used to measure this activity. The assay is also suitable for the specific measurement of (Na+ + K+)-ATPase activities in membranes which contain high levels of other ATPase activities.     

A DIRECT-SPRAY TECHNIQUE FOR THE BIOLOGICAL EVALUATION OF PYRETHRUM-IN-OIL INSECTICIDES FOR USE AGAINST STORED PRODUCT INSECTS IN WAREHOUSES

A direct-spray technique for the biological assay of pyrethrins in Shell oil P31 is described, together with the experiments leading to its adoption. The technique is such that film effects are reduced to the minimum practicable, but it is otherwise as similar as possible to the film technique described by Parkin & Green (1943). Methods for statistical analysis of the results are discussed. For the biological assay of pyrethrins in Shell oil P31, the direct-spray method is slightly superior to Parkin & Green's film method, but both techniques should have their uses in general experimental work.     

Radiochemical assays for adenylate kinase and AMP deaminase using polyethyleneimine-cellulose thin layers

Simple and specific radiochemical asays for adenylate kinase and AMP deaminase activities in crude tissue extracts are described. The radioactive substrate (AMP) is separated from the radioactive product (ADP or IMP) by chromatography on polyethyleneimine-cellulose thin layers. A rapid modification of the adenylate kinase assay is described in which samples of the reaction mixture are transferred directly to the polyethyleneimine-cellulose thin layer.     

Further experiments with digestive enzymes in freshly killed animals

A number of modifications of Freeland's method for the assay of enzymes by radial diffusion in agar are described. Amylase does not obey Fick's Law but methods for producing a standard curve are given. An alternative method of assay is to time the loss of colour in a standard solution of substrate.