A facile,sensitive and quantitative membrane‐binding assay for proteins

Soluble proteins that bind membranes function in numerous cellular pathways yet facile, sensitive and quantitative methods that complement and improve sensitivity of widely used liposomes‐based assays remain unavailable. Here, we describe the utility of a photoactivable fluorescent lipid as a generic reporter of protein‐membrane interactions. When incorporated into liposomes and exposed to ultraviolet (UV), proteins bound to liposomes become crosslinked with the fluorescent lipid and can be readily detected and quantitated by in‐gel fluorescence analysis. This modification obviates the requirement for high‐speed centrifugation spins common to most liposome‐binding assays. We refer to this assay as Proximity‐based Labeling of Membrane‐Associated Proteins (PLiMAP).     

A sensitive assay for allantoin

Allantoin is separated by thin-layer chromatography (TLC) and sprayed with an acidic solution ofp-dimethylaminobenzaldehyde. The yellow color produced is read in a densitometer and compared with that of a standard. The lower limit of quantitation is 0.1 μg per spot (0.5 mg/100 ml). The method can be utilized for the estimation of allantoin in serum, lymph, and urine.     

A sensitive and high-throughput assay to detect low-abundance proteins in serum

The ability to detect antigens immunologically is limited by the affinity of the antibodies and the amount of antigens. We have now succeeded in creating a modular, facile amplification system, termed fluorescent amplification catalyzed by T7 polymerase technique (FACTT). Such a system can detect protein targets specifically at subfemtomolar levels ( approximately 0.08 fM). We describe here the detection of Her2 (also known as Neu) from rodent and human sera. FACTT is adaptable to high-throughput screening and automation and provides a practical method to enhance current ELISAs in medical practice.     

A simple and sensitive assay for dopamine-beta-hydroxylase

A simple, rapid, and highly sensitive radiochemical assay for measuring the activity of dopamine-β-hydroxylase in tissues and serum is described. Enzyme activity is detected by converting [1-¹⁴C]tyramine to [1-¹⁴C]octopamine which is then subjected to periodate cleavage to form [¹⁴C]form-amide. This radiolabeled product is oxidized to ¹⁴CO₂ by addition of permanganate and the ¹⁴CO₂ is trapped and counted. The assay is simple and sensitive, it can linearly detect enzyme in all tissues with a wide range of activity, it uses maximal concentration of substrate, and it requires the addition of only one concentration of EMI to block endogenous inhibitor(s) in different tissues or enzyme concentrations.     

A rapid and sensitive assay for arginase

An assay for arginase is described that uses l-[guanido-¹⁴C]arginine as substrate. Unhydrolyzed arginine is removed in a batch procedure with sulfonate resin and the [¹⁴C]urea product is determined quantitatively in the resin supernatant. The assay requires 5 min and is performed in one tube. The sensitivity is approximately 0.1 munits of arginase. Arginase activities in fetal calf serum and in murine macrophage extract have been determined and the bovine liver enzyme has been used as a reference.     

A sensitive radioenzymatic assay for ATP

We describe a radioenzymatic assay for ATP based on the phosphorylation of radioactively labeled sugar in the presence of the appropriate kinase and further separation from the unreacted sugar using ion-exchange resin. The sensitivity of the method as described here goes down to 10¹² to 10¹⁴ mol of ATP and could be increased using radioactive sugar of higher specific activity.     

A sensitive enzymic assay for benzylpenicillin

A method has been developed for the determination of sodium benzylpenicillin concentrations in the range 3·3–33 μg/ml. 6-Aminopenicillanic acid is released from the benzylpenicillin by the action of the enzyme penicillin acylase and is estimated from its reaction with fluorescamine at pH 4.7-Aminocephalosporanic acid shows a similar trend to 6-aminopenicillanic acid in its reaction at pH 4. The open β-lactam ring form of each compound shows little fluorescence with fluorescamine at pH but shows strong fluorescence in the pH range 7–9. 6-Aminopenicillanic acid and its open β-lactam ring form give different fluorescent responses to increasing volumes of a solution of the fluorigenic agent at pH 7·8. This effect can be used to estimate concentrations in a mixture of the two components providing other amino material is absent.     

A sensitive fluorimetric assay for pyruvate

A sensitive and specific fluorimetric assay for the determination of pyruvate is reported here. This assay is based on the oxidation of pyruvate in the presence of pyruvate oxidase. Hydrogen peroxide generated by pyruvate oxidase reacts with nonfluorescent Amplex Red at a 1:1 stoichiometry to form the fluorescent product, resorufin. The assay is optimized with respect to pH of reaction buffer, enzyme concentration, dye concentration, and the time course. The usefulness of the assay is demonstrated by the accurate measurement of intracellular and extracellular pyruvate concentrations. The limit of detection of the assay is 5 nM.     

A sensitive assay for histone methyltransferase

A method is described for the assay of histone methyltransferase using soluble histones as substrate. The precipitation of the methylated protein on chromatography paper allows for greater sensitivity and more rapid sample processing than have previously been reported. After incubation of the enzyme in the presence of radioisotopically labeled S-adenosyl-l-methionine and soluble rat brain histone, the residual S-adenosyl-l-methionine is removed by extensive washing in 1.1 m trichloroacetic acid. The amount of methyl groups incorporated into histones is measured by liquid seintillation counting. This procedure can probably be used to assay other protein methylases. A comparison is made between this assay and one using chromosomal bound histones as substrates.     

A sensitive assay for chicken glutamyltransferase

The optimal conditions required for measuring chicken glutamyltransferase were examined to obtain a sensitive quantitative assay method for the enzyme. This method has made possible the determination of the enzyme activity in various tissues.     

A sensitive bioluminescence assay for myo-inositol

A specific, sensitive bioluminescence assay for myo-inositol is described employing myo-inositol dehydrogenase linked to a commercial NADH bioluminescence kit. Optimum conditions providing a linear response over 4 orders of magnitude are presented with reproducibility of 6% CV and a sensitivity of 1 pmol. The assay is suitable for the measurement of inositol at low concentrations or in small volumes.     

A simple and sensitive radioreceptor assay for leukotrienes

A simple and sensitive radioreceptor assay (RRA) for leukotrienes (LTs) was developed using a highly specific [³H]leukotriene D₄ (LTD₄) binding to guinea pig lung membrane homogenates. The assay can detect down to 0.15 pmol of LTD₄. The values for fifty percent inhibition of bound [³H]LTD₄ was 1.5 nM for LTD₄, 45 nM for LTC₄ and 24 nm for LTE₄. LTB₄ at 3.0 × 10⁻⁵ M had no effect on [³H]LTD₄ binding. The RRA for LTs in the absence of serine-borate complex was bi-specific for both LTC₄ and LTD₄. However, in the presence of 20 nM serine-borate this method was highly specific for LTD₄. Recovery rate averaged 87.2% after ethanol extraction and evaporation of known amounts of LTD₄. When the radioreceptor assay and radioimmunoassay data for leukotriene levels in the samples were compared to each other, an excellent correlation was observed with a correlation coefficient ‘r’ of 0.992. The assay was also validated by quantitation of LTs released from human granulocytes stimulated with calcium ionophore, A23187. The method is simpler, less expensive, and more specific for LTD₄ than the other methods such as high pressure liquid chromatography and radioimmunoassay and is suitable for routine measurement of either LTD₄ specifically or LTC₄ plus LTD₄ simultaneously in one cell system.     

A simple and sensitive radioreceptor assay for leukotrienes

A simple and sensitive radioreceptor assay (RRA) for leukotrienes (LTs) was developed using a highly specific [3H]leukotriene D4 (LTD4) binding to guinea pig lung membrane homogenates. The assay can detect down to 0.15 pmol of LTD4. The values for fifty percent inhibition of bound [3H]LTD4 was 1.5 nM for LTD4, 45 nM for LTC4 and 24 nM for LTE4. LTB4 at 3.0 X 10(-5)M had no effect on [3H]LTD4 binding. The RRA for LTs in the absence of serine-borate complex was bi-specific for both LTC4 and LTD4. However, in the presence of 20 mM serine-borate this method was highly specific for LTD4. Recovery rate averaged 87.2% after ethanol extraction and evaporation of known amounts of LTD4. When the radioreceptor assay and radioimmunoassay data for leukotriene levels in the samples were compared to each other, an excellent correlation was observed with a correlation coefficient 'r' of 0.992. The assay was also validated by quantitation of Lts released from human granulocytes stimulated with calcium ionophore, A23187. The method is simpler, less expensive, and more specific for LTD4 than the other methods such as high pressure liquid chromatography and radioimmunoassay and is suitable for routine measurement of either LTD4 specifically or LTC4 plus LTD4 simultaneously in one cell system.