Rapid filtration assays for protein kinase C activity and phorbol ester binding using multiwell plates with fitted filtration discs.

In the conventional approach protein kinase activity and phorbol ester binding associated with protein kinase C (PKC) are measured by initially incubating samples in either test tubes or multiwell plates, followed by filtration of the terminated reaction mixture using either a manifold filtration device or a cell harvester. Here we report a method in which both the incubations and filtrations necessary for the determination of either protein kinase activity or phorbol ester binding are carried out in the same multiwell plate with fitted filtration discs made of polyvinylidene difluoride (Durapore membrane). Due to the very low binding of protein to these filters, there is no interference caused by these filters during the incubation period of the assays. The drawback with these filters compared to commonly used cellulose acetate membrane filters is that they retain less of the phosphate acceptor substrate histone H1 (only 15%) if filtered and washed with standard 5% trichloroacetic acid. However, this can be overcome by increasing the trichloroacetic acid concentration to 25% during filtration. For phorbol ester binding determinations, the samples are incubated with [3H]phorbol 12,13-dibutyrate in the microwells, the ligand bound PKC is adsorbed onto DEAE-Sephadex beads, and the beads then are filtered and washed in the same microwells. Furthermore, this multiwell filtration approach can also be adopted to previously described cytosolic phorbol ester receptor assays, which have the broader conditions for optimal binding to receptors. Durapore membrane filters are found to work well for punching into scintillation vials and there is complete recovery of the radioactivity retained with the filters. In the protein kinase assay the background radioactivity is very low (< 200 cpm) and in the phorbol ester binding assay the nonspecific binding is less than 1%. Thus, these low background values result in at least a fourfold increase in sensitivity for these assays. Since the incubations and filtrations are carried out in the same well without any transfer of the sample, the coefficient of variation in multiple determinations is found to be low. Furthermore, this method is rapid and more convenient for analyzing a larger number of samples than conventional methods which use test tubes, and it is less expensive to set up compared to the automated methods that use a cell harvester.     

Automation of a chloramphenicol acetyltransferase assay

Accurate quantification of chloramphenicol acetyltransferase (CAT) enzyme activity in a large number of samples has been achieved through robotization of a CAT assay on a laboratory workstation (Biomek 1000). The basic principle of this CAT assay relies on the selective diffusion of [3H]acetylchloramphenicol into a water-immiscible liquid scintillation cocktail. This methodology gives unique characteristics to this robotized protocol by allowing complete control over the kinetics of the CAT enzymatic reaction which is a critical parameter in the CAT assay. Thus it has been possible to optimize the CAT assay for every processed sample, through real time monitoring of the enzymatic reaction, and to achieve maximum accuracy in CAT quantification. Moreover the sensitivity of this automated assay is high (detection threshold; 10(-4) CAT unit), and the sample processing is fast (approximately 125 samples per hour). Compared to other CAT assay protocols currently used, our robotized technique offers major advantages in terms of CAT quantification, and sets new standards for CAT assay productivity.     

A simple quantitative assay for chloramphenicol acetyltransferase by direct extraction of the labeled product into scintillation cocktail.

A simple, rapid, sensitive, quantitative, and inexpensive assay for chloramphenicol acetyltransferase (CAT) is described. The assay is based on the direct extraction of the products of the reaction into toluene-based liquid scintillation cocktail. The assay is carried out in 7-ml scintillation vials using 1 mM chloramphenicol and either 100 microM acetyl-CoA and 0.1 microCi of [3H]acetyl-CoA or 1 mM acetyl-CoA and 0.5 microCi of [3H]acetyl-CoA. After incubation, the reaction is terminated with 0.5 ml of 0.1 M sodium borate-5 M NaC, pH 9. The acetylchloramphenicols are extracted with 5 ml of 0.4% 2,5-diphenyloxazole-0.005% 1,4-bis(5-phenyloxazol-2-yl)benzene in toluene by a 30-s shaking. After a short centrifugation to clarify the layers, the vials are counted in a liquid scintillation counter. Extracted products are stable in the organic layer. Under these conditions, nearly 100% extraction of acetylchloramphenicols is shown using nonlabeled compounds and spectrophotometric methods. Using pure enzyme in the assay, linearity of activity with enzyme concentration, time, and temperature of incubation is demonstrated. Assays may even be carried out at 60 degrees C, where the enzyme activity is 3.4-fold higher than that at 23 degrees C. The increase in enzyme activity with increasing temperature is due to the increased formation of predominantly 3-acetyl and 1-acetylchloramphenicols and not to 1,3-diacetylchloramphenicol. The present assay compared very well with the standard assay using [14C]chloramphenicol and TLC. Using this assay, we measured quantitatively the CAT activity in extracts of pSV2-CAT-transfected CV-1 cells in 10 min and NIH 3T3 cell extracts in 60 min at 60 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)     

A radioenzymatic assay for catecholamines and dihydroxyphenylacetic acid.

Picogram quantities of the catecholamines, dopamine, norepinephrine, and epinephrine, and the dopamine metabolite, dihydroxyphenylacetic acid, can be measured in tissue or plasma samples utilizing a rapid radioenzymatic procedure. The catechols are converted to their ³H-methylated derivatives (3-methoxytyramine, normetanephrine, metanephrine and homovanillic acid, respectively) by the enzyme catechol-O-methyltransferase with ³H-S-adenosylmethionine serving as the ³H-methyl donor. Following the enzymatic reaction, unreacted ³H-S-Adenosylmethionine is removed by precipitation and the reaction products are separated by thin layer chromatography on silica plates. The areas corresponding to the ³H-methylated derivatives are scraped into scintillation vials, eluted with aqueous buffer, extracted into nonpolar scintillation cocktail, and counted by liquid scintillation spectrometry. Using the standard assay procedure described here, over 100 tubes can be assayed in a single day with a sensitivity of 15–25 pg for all compounds measured. With the application of additional procedures, as little as 1 pg norepinephrine and epinephrine and 5–10 pg dopamine and dihydroxyphenylacetic acid can be quantified in a single sample.     

Herpes simplex virus thymidine kinase enzymatic assay in transient transfection experiments using thymidine kinase-deficient cells.

An enzymatic assay for herpes virus simplex type 1 thymidine kinase (HSV-TK) that was sensitive enough to quantitate intracellular levels of enzyme transiently expressed after transfection of HSV-TK vectors into TK-deficient cells using the DNA-calcium phosphate coprecipitation technique is described. TK activity in extracts of transfected cells was determined by binding of [methyl-3H]thymidylate product to thin layers of polyethyleneimine (PEI)-impregnated cellulose. The assay used high-specific-activity [methyl-3H]thymidine as substrate, which required removal of anionic material on a column of PEI-cellulose to enhance the signal-to-noise ratio. The assay was linear over a wide range with respect to the amount of HSV-TK plasmid transfected or content of HSV-TK enzyme in cell extracts. To validate the assay in transient expression experiments, HSV-TK and chloramphenicol acetyltransferase (CAT) plasmids were cotransfected into NIH/3T3 tk- fibroblasts. Transient TK and CAT levels were concordant in cell extracts prepared from replicate plates of transfected cells. Normalizing the transient TK activity for CAT activity from the cotransfected "internal standard" CAT plasmid improved precision significantly, reducing the sample-to-sample coefficient of variation from 41 to 19%. CAT normalization reduced experimental variability mostly by correcting outlying results in transfection efficiency. The HSV-TK reporter gene system based on TK enzymatic assay was thus subject to experimental variation similar to that of the well-established CAT reporter function, demonstrating its utility in transient gene expression analysis.     

An economical system for liquid scintillation counting

Small glass shell vials (12 × 35 mm minivials), containing 2.0 ml of a dioxane-based scintillation solution plus a ¹⁴C-labeled sample, were placed in a conventional glass, 20-ml count vial and assayed in a scintillation spectrometer. Statistical comparison of counts recorded from ¹⁴C samples prepared both in the minivial system and conventional 20-ml count vials indicated that the two systems were equivalent with sample volumes of 10 and 100 μliters (1600-cpm solution) and 10 μliters (60-cpm solution). Conventional 20-ml glass or plastic count vials were both acceptable as containers for the minivials.There were no significant differences in the counts from samples in minivials placed on-center and off-center in the container vial. Cost per sample was reduced from 24.8 cents (conventional glass vials) to 4.7 cents (minivial system).     

Protection of histones isolated from elastase-treated mouse epidermis

Small glass shell vials (12 × 35 mm minivials), containing 2.0 ml of a dioxane-based scintillation solution plus a ¹⁴C-labeled sample, were placed in a conventional glass, 20-ml count vial and assayed in a scintillation spectrometer. Statistical comparison of counts recorded from ¹⁴C samples prepared both in the minivial system and conventional 20-ml count vials indicated that the two systems were equivalent with sample volumes of 10 and 100 μliters (1600-cpm solution) and 10 μliters (60-cpm solution). Conventional 20-ml glass or plastic count vials were both acceptable as containers for the minivials.There were no significant differences in the counts from samples in minivials placed on-center and off-center in the container vial. Cost per sample was reduced from 24.8 cents (conventional glass vials) to 4.7 cents (minivial system).     

Microdetermination of inorganic sulfate using thin-layer plates

Inorganic sulfate was precipitated on cellulose thin-layer plates with the radioactive reagent, ¹³³BaCl₂. Excess reagent was removed by repeated washings with an acidic BaCl₂ solution. The residual activity was transferred to vials by cutting out the point of application and its immediate surroundings. Counting was performed in a scintillation well γ-counting system. The concentration-activity curve was linear.     

A highly sensitive, mixed-phase assay for chloramphenicol acetyltransferase activity in transfected cells

We describe a simple, rapid yet extremely sensitive assay for chloramphenicol acetyltransferase (CAT) activity in extracts from transfected eukaryotic cells. Using our modified reaction conditions and the mixed-phase assay, less than 0.000010 unit of CAT activity in transfected cells can be reliably detected. The mixed-phase assay is based on the inability of the polar [3H]-acetyl-Coenzyme A (CoA) substrate to partition out of a urea containing aqueous phase into the nonpolar scintillation fluor, while the [3H]chloramphenicol reaction products partition into the toluene scintillation fluor and are quantitated by scintillation counting. The increased sensitivity of this assay is due to the optimization of the acetyl-CoA concentration, to a urea-containing aqueous phase which lowers the assay background, and to the use of extract blanks. The mixed-phase assay is simpler, is quantitative, uses less costly substrates, and is far more sensitive than the most widely used CAT assays, which require solvent extraction followed by thin-layer chromatography to separate the unreacted substrate from product.     

Two continuous coupled assays for ornithine-δ-aminotransferase

We have developed two new continuous coupled assays for ornithine-δ-aminotransferase (OAT) that are more sensitive than previous methods, measure activity in real time, and can be carried out in multiwell plates for convenience and high throughput. The first assay is based on the reduction of Δ¹-pyrroline-5-carboxylate (P5C), generated from ornithine by OAT, using human pyrroline 5-carboxylate reductase 1 (PYCR1), which results in the concomitant oxidation of NADH (nicotinamide adenine dinucleotide, reduced form) to NAD⁺ (nicotinamide adenine dinucleotide, oxidized form). This procedure was found to be three times more sensitive than previous methods and is suitable for the study of small molecules as inhibitors or inactivators of OAT or as a method to determine OAT activity in unknown samples. The second method involves the detection of l-glutamate, produced during the regeneration of the cofactor pyridoxal 5’-phosphate (PLP) of OAT by an unamplified modification of the commercially available Amplex Red l-glutamate detection kit (Life Technologies). This assay is recommended for the determination of the substrate activity of small molecules against OAT; measuring the transformation of l-ornithine at high concentrations by this assay is complicated by the fact that it also acts as a substrate for the l-glutamate oxidase (GluOx) reporter enzyme.     

The effect of sample composition and vial type on Ĉerenkov counting in a liquid scintillation counter

The effect of sample vial type and sample composition on the ?erenkov count rate detected from ³²P and ³⁶Cl was studied using a liquid scintillation counter. When counting was done in the noncoincident mode, glass vials allowed higher counting efficiency than plastic vials. In the coincident mode light scattering caused by polyethylene and polyproplyene vials allowed higher counting efficiency than glass vials. Highest coincident counting efficiency was from plastic minivials in a glass carrier vial. Increased solute concentration in samples caused increased counting efficiency due to changes in the refractive index of the solution. This can cause significant counting efficiency changes with no sample channel ratio change in density gradient fractions. The use of wavelength shifters is shown to be inappropriate when the sample pH varies, as this can change the fluorescent properties of the shifters and thereby the observed count rate.     

RARβ在胃癌细胞生长调节中的作用

为探讨 RARβ受体介导全反式视黄酸 ( ATRA)抑制胃癌细胞生长的作用机理 ,用 Northern印迹测定 RARβ m RNA表达水平 ,脂质体介导的转染方法将含有 RARβ基因的表达载体转染MKN- 45细胞并稳定表达 ,MTT和软琼脂集落形成等实验测定细胞生长速率和生长状态 ,氯霉素乙酰转移酶活性 ( CAT)测定视黄酸应答元件βRARE的转录活性以及 AP- 1 ( activator protein- 1 )活性 .RARβ在 ATRA敏感细胞株 MGC80 - 3、BGC- 82 3和 SGC- 790 1中表达 ,而在 ATRA抗性细胞株 MKN- 45中不表达 .当 RARβ基因转染 MKN- 45细胞时 ,细胞变为 ATRA敏感 ,由此导致ATRA抑制 MKN- 45细胞生长和软琼脂集落形成 .ATRA可以加强诱导 MGC80 - 3、BGC- 82 3和SGC- 790 1细胞βRARE的转录活性 ,但对 MKN- 45细胞影响不大 ,不能抑制细胞 AP- 1活性 .当RARβ基因转染 MKN- 45细胞后 ,ATRA则能够诱导细胞 βRARE的转录活性 ,并抑制细胞的 AP-1活性 .RARβ表达与 ATRA抑制胃癌细胞生长密切相关 .ATRA诱导 βRARE转录活性和抑制AP- 1活性可能是其调控胃癌细胞生长的机制之一 .     

A Rapid and Simple Method for Assaying Interferon1

A new procedure of assaying interferon (IF) has been developed. Cell suspension was dispensed into liquid scintillation counting vials together with IF sample. During an overnight incubation, the cells adhered sufficiently to the bottom of the vials and all the subsequent procedures were carried out without transfer of the cells from the vials. Vesicular stomatitis virus was inoculated and virus-specific RNA was labeled by adding ³H-uridine and actinomycin D to the medium. Incubation was terminated prior to completion of a single-step growth of the virus and radioactivity of the labeled cells in each vial was determined. The reciprocal of the IF dilution which reduced the radioactivity in viral RNA by 50% was taken as the titer. The present procedure consists of simple manipulations and can be completed within 24 hr. Furthermore, it is quite reproducible and gives a titer almost identical to that obtained by the conventional plaque-reduction dose method. The procedure can be applied to mouse L cells, rabbit RK-13 cells and human FL cells, without modification.     

Liquid scintillation counting of radioactive monomeric and polymeric carbohydrates on paper chromatograms

A simple, improved scintillation counting procedure was developed for the assay of radioactive mono- and polysaccharides on paper chromatograms. Segments of chromatograms are placed in scintillation vials and soaked in water to completely elute the carbohydrate before addition of Aquasol, a xylene-based scintillation fluid. The resulting water-Aquasol solution is counted in a liquid scintillation counter. Evaluation of numerous experimental variables revealed optimal conditions for complete elution of mono- and polysaccharides with water before counting in Aquasol.The water elution-Aquasol procedure allows water-soluble substances (¹⁴C- and ³H-labeled) on paper to be assayed with increased accuracy and sensitivity (three- to fivefold improvement in counting efficiency of tritiated samples). The simplicity of the procedure allows entire radiochromatograms to be assayed readily.     

Counting of p32 in plant tissues using the Cerenkov effect

Summary A procedure for counting p³² in plant tissues is presented. The method, based on the use of Cerenkov radiation, involves practically no sample preparation. Plant tissue are placed into vials containing water or hexane and counted with a liquid scintillation counter. Counts obtained, using this procedure were found to be linearily related to that obtained with a G.M. tube. The counting efficiency was, however, higher with the proposed method. The use of hexane is advantageous if leakage of p³² from the tissue is possible, or when higher counting efficiency is desireable. The use of different liquids may also enable a discriminative count of different beta emitters. As suggested recently⁸ use of wavelength shifter may further increase efficiency of counting Cerenkov radiation.