Dot assay for neomycin phosphotransferase activity in crude cell extracts

A dot assay for determining neomycin phosphotransferase (NPT II) activity in crude cell extracts has been developed. The assay provides for the rapid screening of large numbers of cell cultures generated in gene transformation experiments using NPT II as a dominant selectable marker. Currently, the commonly used procedure for NPT II assay employs a time-consuming electrophoretic protein separation step to eliminate a positive interference resulting from putative protein kinase activities present in crude cell extracts. The dot method we have developed is based upon the ability of nitrocellulose membrane to eliminate that positive interference without a prior protein separation step. It provides a sensitive, reproducible, and significantly more convenient and rapid means of screening large numbers of cell extracts in order to distinguish cultures producing high levels of NPT II from those that do not.     

A new sensitive method for qualitative and quantitative assay of neomycin phosphotransferase in crude cell extracts

A general method is described for the detection and quantification of low amounts of neomycin phosphotransferase in crude cell extracts. The assay is based on the electrophoretic separation of the enzyme from other interfering proteins and detection of its enzymatic activity by in situ phosphorylation of the antibiotic kanamycin. Both kanamycin and [γ³²P]ATP acting as substrates are embedded in an agarose gel placed on the polyacrylamide gel containing the separated proteins. After the enzymatic reaction, the phosphorylated kanamycin is transferred to P81 phosphocellulose ion exchange paper and the radiolabeled kanamycin is visualised by autoradiography. With this method 1 ng of active enzyme can easily be detected. Both prokaryotic and eukaryotic cell extracts can be examined, and changes in the size of enzymatically active proteins can be determined.     

Assay of neomycin phosphotransferase activity in cell extracts.

A simple method for the measurement of neomycin phosphotransferase (NPT) activity in crude extracts of eukaryotic cells is described. This method is based on the elimination of interfering phosphorylated proteins by using phenol-chloroform extraction. This solution phase assay allows the detection of greater than or equal to 0.01 ng of NPT in the crude cell extract. Rapid screening of a large number of cell cultures generated in gene-transfer experiments, using NPT as a selective marker, is made possible by this simple technique. Further, the promoter strength of vector constructs used in gene therapy may also be estimated by this procedure.     

Measurement of hygromycin B phosphotransferase activity in crude mammalian cell extracts by a simple dot-blot assay.

Hygromycin B (Hy) resistance, encoded by the prokaryotic gene hph, is commonly used as a dominant selectable marker for gene transfer experiments in mammalian cells. We describe a simple, quantitative dot-blot assay for measuring the activity in crude mammalian cell extracts of Hy phosphotransferase, the product of the hph gene. The assay shows no cross interference with substrates for neomycin phosphotransferase II, the product of the commonly used marker gene neo; hph and neo may thus be useful as a set of two non-interfering selectable marker and reporter genes for gene transfer experiments in mammalian cells.     

A quantitative assay for neomycin phosphotransferase activity in plants

A sensitive, simple, and quantitative assay for determining neomycin phosphotransferase (NPT) activity in plant cell extracts is described. The procedure retains the simplicity of previously published methods, yet offers up to a 140-fold increase in sensitivity. This increase is due to (1) the addition of bovine serum albumin (BSA) to the assay mixture, (2) desalting of crude maize extracts to remove a low-molecular-weight inhibitor of the enzyme, and (3) use of a different extraction buffer and an improved extraction procedure to liberate more enzyme from the cells. This method has been used successfully to detect and quantitate both stable and transient expression of NPT in transgenic tobacco and maize tissue.     

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.     

A rapid immunoprecipitation assay for neomycin phosphotransferase II expression in transformed bacteria and plant tissues

Anti-kanamycin antibodies produced in rabbits, following coupling of the antibiotic to bovine serum albumin, were used to immunoprecipitate radioactively labelled phosphorylated kanamycin from transformed bacterial or plant extracts in a novel assay system, for the detection of neomycin phosphotransferase II (NPTII) activity. Radioactive counts in the immunoprecipitated pellet give a semiquantitative measure of the kanamycin phosphorylation and hence the amount of NPTII activity. This assay is sensitive, uses very small amounts of radioactivity, and is very rapid, allowing many samples to be processed within a few hours. Immunoprecipitated counts from reactions with bacteria carrying a kanamycin resistance gene or from tobacco and Brassica napus plants transformed with NPTII gene-containing vectors were consistently higher than counts from nontransformed controls. Results obtained with this assay correlate well with those from the previously described gel overlay and dot-blot assays, but can be obtained in an appreciably shorter time frame.     

Evaluation of an ELISA assay for rapid detection and quantification of neomycin phosphotransferase II in transgenic plants

Neomycin phosphotransferase II (neo) is a selectable marker gene used extensively in plant transformation experiments. Here we evaluate immunological detection of its gene product (NPTII) as an alternative to widely used radioactive assays. We have taken a commercially available non-radioactive NPTII Enzyme linked-Immunosorbant Assay (ELISA) kit, modified the protocol for application to plant tissues, and used it to quantify levels of NPTII protein in transformed plants. The ELISA proved safe, economical and convenient to reliably screen and quantify NPTII protein in large numbers of plant samples. The sensitivity of the ELISA for NPTII detection in tobacco plants is at least an order of magnitude greater than a widely used radioactive gel assay. Using three replicates per sample, standard errors are low and the assay is highly reproducibleover time for tissue-cultured tobacco. However, background readings varied with plant species, and also with plant age for untransformed glasshouse-grown tobacco. It is therefore essential to ensure that untransformed controls are closely matched to test plant.     

A new neomycin phosphotransferase II solid phase assay in combination with polyacrylamide sodium dodecylsulphate gel electrophoresis

A new general method for the determination of neomycin phosphotransferase (NPT) II (EC 2.7.1.95) activity in cell extracts after separation in SDS-polyacrylamide gels is described. The enzymatic activity of NPT II is restored after SDS-polyacrylamide gel electrophoresis by incubating the gel for 3 h (20 mM Tris-HCl buffer, pH 7.4). The enzymatic activity is determined by in situ phosphorylation of aminoglycoside antibiotics bound to solid supports and brought into direct contact with the gel surface. A novel, mechanically stable, negatively charged matrix was synthesized for use in this solid phase enzyme assay and compared to phosphocellulose and carboxymethylcellulose paper. This new method allows the easy and exact determination of the molecular weight of any fusion protein with NPT II by assaying the position of the enzymatic activity in the gel and a consecutive immunological reaction following protein transfer onto nitrocellulose membranes.     

Fluorimetric urease inhibition assay on a multilayer microfluidic chip with immunoaffinity immobilized enzyme reactors

We fabricated a three-layer polydimethylsiloxane (PDMS)-based microfluidic chip for realizing urease inhibition assay with sensitive fluorescence detection. Procedures such as sample prehandling, enzyme reaction, reagent mixing, fluorescence derivatization, and detection can be readily carried out. Urease reactors were prepared by adsorption of rabbit immunoglobulin G (IgG) and immunoreaction with urease-conjugated goat anti-rabbit IgG. Acetohydroxamic acid (AHA) as a competitive inhibitor of urease was tested on the chip. Microfluidically generated gradient concentrations of AHA with substrate (urea) were loaded into urease reactors. After incubation, the produced ammonia was transported out of reactors and then reacted with o-phthalaldehyde (OPA) to generate fluorescent products. Urease inhibition was indicated by a decrease in fluorescence signal detected by microplate reader. The IC₅₀ value of AHA was determined and showed good agreement with that obtained in microplate. The presented device combines several steps of the analytical process with advantages of low reagent consumption, reduced analysis time, and ease of manipulation. This microfluidic approach can be extended to the screening of inhibitory compounds in drug discovery.     

A quantitative solid-phase assay for identifying radiolabeled glycosaminoglycans in crude cell extracts

Extraction of radiosulfate-labeled cell layers in denaturing urea and nonionic detergent allows the quantitative binding of GAG-containing materials from up to 96 discrete samples to a single cationic nylon blot. Free sulfate and/or sulfated lipids fail to bind. Washing the blot with differential salt concentrations discriminates between native proteoglycans and free glycosaminoglycan chains or fragments. In addition, chondroitin sulfates and heparan sulfate are identified either by prior digestion with chondroitin ABC or AC lyase, as generated disaccharides fail to bind to the blot, or by treatment of the entire blot with nitrous acid following binding. Similarly, heparan sulfate can be identified on chromatograms or Western transfers from polyacrylamide gel electrophoresis by autoradiography before and after treatment of the blot with nitrous acid.     

A bifunctional fusion between beta-glucuronidase and neomycin phosphotransferase: a broad-spectrum marker enzyme for plants.

We have used an in vivo selection approach to isolate a gene encoding a bifunctional fusion peptide between Escherichia coli beta-glucuronidase (GUS) and neomycin phosphotransferase II (NPT-II) from transposon Tn5 in the NH2-GUS::NPT-II-COOH configuration. The fused gene is predicted to encode a fusion peptide 885 amino acids long, and was shown in E. coli to synthesize a 97-kDa GUS+ NPT-II+ gene product. Gel-filtration chromatography suggested that, while the native GUS may be active as a dimer and NPT-II as a monomer, the elution profile of the fusion protein is consistent with that of a trimer. The fusion marker has been produced and defined in transgenic Nicotiana tabacum plants, where both the chimeric gene and the gene product were stable. The bifunctional gene enabled direct KmR selection at the callus stage and enzymatic or histochemical assessment of the steady-state production of GUS activity in regenerated plants. In addition to allowing structure-function determination for the GUS and NPT-II domains of the fusion peptide, the gus::npt-II gene simplifies vector constructs where both marker domains are desired.     

Modified ELISA for the detection of neomycin phosphotransferase II in transformed plant species

 Identifying transformed plant lines carrying the antibiotic resistance marker gene, neomycin phosphotransferase II, requires a more definitive test than the ability of the plant to grow on kanamycin. Although a number of alternative assays have been described, most are cumbersome, time consuming and/or require the use of radioisotopes. This report describes an ELISA for the detection of the neomycin phosphotransferase II enzyme in transformed plant tissue. The ELISA utilises commercially available antibodies and provides a number of advantages, including an extremely low background, a reduction in the amount of tissue required for testing, and semi-quantitative data on neomycin phosphotransferase II gene expression. This method has been applied successfully to a number of independently transformed lines in nine plant species. Received: 4 January 1999 / Accepted: 17 April 1999     

An improved method for determining the activity of benzoyl-arginine p-nitroanilide-hydrolyzing enzyme in crude tissue extracts

The method described in this communication is sensitive and allows the direct determination in crude tissue extracts of the enzyme that hydrolyzes α-N-benzoyl-dl-arginine p-nitroanilde (Bz-Arg-NPhNO₂). It may be useful in determining cathepsin B activity in crude enzyme preparations when the reaction mixture is incubated with an inhibitor of trypsinlike enzymes which are also capable of hydrolyzing Bz-Arg-NPhNO₂.     

A simple spectrophotometric assay for fumarate hydratase in crude tissue extracts

A procedure is described for assaying fumarate hydratase by coupling malate formed from fumarate to NADP⁺ reduction via NADP malic enzyme. The procedure is much more sensitive than existing assay methods and cireumvents problems particularly associated with the use of these methods for determining fumarate hydratase in crude tissue extracts.