A sensitive fluorometric assay for tissue transglutaminase

We have devised a highly sensitive fluorometric well plate assay for tissue transglutaminase that is suitable for multiple kinetic analyses/high-throughput screening of chemical inventories for inhibitors of this enzyme. The procedure measures the rate of fluorescence enhancement (lambda(exc) 260 nm, lambda(em) 538 nm) when 1-N-(carbobenzoxy-l-glutaminylglycyl)-5-N-(5'N'N'-dimethylaminonaphthalenesulfonyl)diamidopentane (glutaminyl substrate) is cross-linked to dansyl cadaverine (amine substrate). The assay procedure can be used to measure the activity of as little as 60 microU of purified guinea pig liver tissue transglutaminase (4.2 ng or 54 fmol of enzyme).     

Multiplexed protein detection by proximity ligation for cancer biomarker validation

We present a proximity ligation-based multiplexed protein detection procedure in which several selected proteins can be detected via unique nucleic-acid identifiers and subsequently quantified by real-time PCR. The assay requires a 1-microl sample, has low-femtomolar sensitivity as well as five-log linear range and allows for modular multiplexing without cross-reactivity. The procedure can use a single polyclonal antibody batch for each target protein, simplifying affinity-reagent creation for new biomarker candidates.     

A simple, rapid, and sensitive procedure for the assay of endoproteases using Coomassie brilliant blue G-250

A rapid colorimetric method for the assay of proteolytic enzymes based on the binding of Coomassie brilliant blue G-250 to unhydrolyzed protein substrate is described. Considerable assay time is saved since the method does not require the separation of the hydrolyzed products from the undergraded protein substrate. The procedure is applicable to crude as well as purified preparations of various proteolytic enzymes and compares well with the procedure of M. L. Anson.     

A microtiter-based assay for protein kinase activity suitable for the analysis of large numbers of samples, and its application to the study of Drosophila learning mutants

We have developed a microtiter-based assay for protein kinase activity which depends on the immobilization of substrate proteins to nitrocellulose. The technique makes use of a filtration manifold, allowing as much as a 10-fold increase in efficiency as compared to other protein kinase assays. We have used this assay to measure cAMP-dependent protein kinase (PKA) in Drosophila learning and memory mutants, with exogenous and endogenous substrates. An alteration was found in the affinity of PKA in the mutant turnip. The procedure should be useful for rapid screening of mutants and drugs and could be adapted to additional types of protein kinases as well as protein phosphatases.     

Modified kit procedure for the kinetic assay of ethanol

We have examined the utility of a commercial kit procedure for the determination of ethanol (EtOH), based upon its enzymatic oxidation and the concurrent production of NADH, monitored by photometry at 340 nm. We found that the equilibrium production of NADH is not stoichiometric with respect to initial ethanol concentration, and that with this procedure, the calibration curve for end-point assay of ethanol is linear only for very dilute solutions. Likewise, the kinetic assay of ethanol using the kit procedure is limited to very dilute samples (i.e., concentration in the reaction mixture of ?2.3 mg EtOH/liter). We describe a simple modification of the kit procedure which makes it amenable to the precise kinetic assay of up to 150 mg EtOH/liter in the reaction mixture. This increase in the dynamic range for kinetic assay of ethanol results form the use of hydrazine both as a trapping agent for the acetaldehyde reaction product and as a competitive inhibitor of alcohol dehydrogenase enzyme.     

A rapid colorimetric assay for gamma-glutamyl hydrolase (conjugase).

A simple procedure for the measurement of gamma-glutamyl hydrolase (conjugase) activity is described. Glutamic acid released from pteroylpenta-gamma-glutamate by hog kidney and chicken pancreas conjugases was quantitated using the dye 4,4'-bis(dimethylamino)benzophenone hydrazone. The procedure involves hydrolysis of the folylpoly-gamma-glutamate substrate by conjugase, conversion of glutamate to alpha-ketoglutarate by L-glutamate dehydrogenase and colorimetric measurement of the BDBH derivative of alpha-ketoglutarate. The release of as little as one nmol of glutamic acid from the substrate can be measured by this procedure, which is well suited for the assay of a variety of conjugase preparations. In addition, the method should provide a general assay for the enzymatic hydrolysis of various folate and antifolate polyglutamates.     

Application of glutaraldehyde to in-cell Western assay for normalization

Normalization is essential to the in-cell Western (ICW) assay, a near-infrared immunocytoblot for protein analysis. Here we report that cells reacted with glutaraldehyde fluoresced in the near-infrared region of the spectrum, and the intensity of fluorescence was directly proportional to cell number over a range from 3125 to 100,000 cells per well. We took advantage of this property to develop a method for quantification of cells, and applied it to the ICW assay for normalization. The application of glutaraldehyde may make the ICW assay more popular due to the reduced cost and simplified procedure.     

Diphenylamine-colorimetric method for DNA assay: a shortened procedure by incubating samples at 50 degrees C

We describe a modified procedure of the diphenylamine-colorimetric method for assay of DNA that is shorter than the procedure described by Burton. This improvement is achieved by raising the sample incubation temperature to 50 degrees C. The higher temperature hastens and stabilizes the diphenylamine reaction with DNA so that absorbances of samples can be measured as early as 3 h after the reaction is started. This shortened assay is able to detect as little as 3 micrograms of calf thymus DNA. This method is suitable for measuring DNA content of epidermal tissue.     

Large-scale preparation and biological activity of recombinant human parathyroid hormone

Human parathyroid hormone (hPTH) has been bacterially expressed in bioreactors as cro-β-galactosidase-hPTH fusion protein. We have developed a large-scale purification scheme that exploits the pH-dependent differential solubility of hPTH and a two-step Chromatographie procedure. We demonstrate that in a number of assay systems, the recombinant material obtained by this procedure is biologically active.     

Comparison between different markers for sperm quality in the cat: Diff-Quik as a simple optical technique to assess changes in the DNA of feline epididymal sperm

The majority of wild felids, as well as some domestic cats, have low sperm concentration in their ejaculates, and a high proportion of abnormal spermatozoa. We have employed several possible semen quality markers to further characterize cat epididymal sperm. Methods included possible apoptotic reporters, such as the annexin V assay to monitor exposure of phosphatidylserine (PS) on the outer leaflet of the plasma membrane, as well as cell integrity; and the TUNEL assay to quantify DNA breaks. Sperm surface ubiquitination, another putative marker of sperm quality, was also monitored. The annexin V assay revealed a high percentage of sperm with PS exposure, and the TUNEL assay pointed to high levels (13+/-12%) of sperm with DNA breaks. Correlations were found between apoptotic markers (but not ubiquitination) and semen parameters. In parallel to this analysis, cat sperm morphology was evaluated using the Diff-Quik optical stain, which has been used in human reproduction laboratories. Several types of abnormalities could be characterized with this method. Remarkably, head staining abnormalities detected using the Diff-Quik staining method were strongly correlated with, and could accurately predict, sperm DNA defects detected in the same sample using the TUNEL assay. We therefore suggest that sperm morphology analysis using Diff-Quik could be used in field conditions to assess sperm status, due to the simplicity of the procedure and the equipment involved.     

A microassay for proteolytic activity

A quantitative procedure for measuring proteolytic activity, utilizing azoalbumin as substrate, has been developed for use in microtiter plates. An enzyme-linked immunosorbent assay reader is used to measure absorbance. The procedure is sensitive, as well as being both rapid and economical. It is particularly convenient for measuring large numbers of samples, such as fractions from column chromatography.     

A collagenase assay using [3H-methyl]collagen

A new assay procedure for collagenase is presented. Highly radioactive substrate is prepared by methylation of native collagen. The ³H-labelled protein is readily attacked by bacterial as well as by mammalian collagenase and resistant to other proteinases. The sensitivity of this assay is higher than that of the enzymic methods hitherto available.     

In-gel protein phosphatase assay using fluorogenic substrates

We developed a method for the detection of phosphatase activity using fluorogenic substrates after polyacrylamide gel electrophoresis. When phosphatases such as Ca²⁺/calmodulin-dependent protein kinase phosphatase (CaMKP), protein phosphatase 2C (PP2C), protein phosphatase 5 (PP5), and alkaline phosphatase were resolved by polyacrylamide gel electrophoresis in the absence of SDS and the gel was incubated with a fluorogenic substrate such as 4-methylumbelliferyl phosphate (MUP), all of these phosphatase activities could be detected in situ. Although 6,8-difluoro-4-methylumbelliferyl phosphate (DiFMUP) as well as MUP could be used as a fluorogenic substrate for an in-gel assay, MUP exhibited lower background fluorescence. Using this procedure, several fluorescent bands that correspond to endogenous phosphatases were observed after electrophoresis of various crude samples. The in-gel phosphatase assay could also be used to detect protein phosphatases resolved by SDS-polyacrylamide gel electrophoresis. In this case, however, the denaturation/renaturation process of resolved proteins was necessary for the detection of phosphatase activity. This procedure could be used for detection of renaturable protein phosphatases such as CaMKP and some other phosphatases expressed in cell extracts. The present fluorescent in-gel phosphatase assay is very useful, since no radioactive compounds or no special apparatus are required.     

Improved band shift assay for the simultaneous analysis of protein-DNA interactions and enzymatic functions of DNA polymerases.

A simple method to assay the major properties of DNA polymerases such as template binding, polymerase and exonuclease activities in one step is exemplified with the DNA polymerases of E. coli, bacteriophage T4 and herpes simplex virus. Combining the advantages of the band-shift assay with the resolving power of polyacrylamide gradient gel electrophoresis, the procedure is particularly useful for a rapid functional analysis of mutant polymerases as well as inhibitors of DNA replication.     

Monitoring the active transport of efflux pumps after their reconstitution into proteoliposomes: caveats and keys

There is an acute need for a functional assay allowing the investigation of efflux pumps. A dedicated procedure was previously developed, but although it was unambiguous, it suffered from a lack of reproducibility. We describe an optimization of the procedure that makes the assay much more robust.     

Analysis of RhoA and Rho GEF activity in whole cells and the cell nucleus

We have recently shown that a fraction of the total cellular pool of the small GTPase RhoA resides in the nucleus, and that the nuclear guanine nucleotide exchange factor (GEF) Net1 has a role in the regulation of its activity. In this protocol, we describe a method to measure both the activities of the nuclear pools of RhoA and Rho GEFs. This process required the development of a nuclear isolation protocol that is both fast and virtually free of cytosolic and membrane contaminants, as well as a redesign of existing RhoA and Rho GEF activity assays so that they work in nuclear samples. This protocol can be also used for other Rho GTPases and Rho GEFs, which have also been found in the nucleus. Completion of the procedure, including nuclear isolation and RhoA or Rho GEF activity assay, takes 1 h 40 min. We also include details of how to perform a basic assay of whole-cell extracts.     

An assay for acidic peptide substrates of protein kinases

The assay of acidic peptides as substrates for protein kinases has not been as easy to perform as testing basic peptides or polypeptides. We have developed a simple, rapid, and cost-effective procedure that allows the design and testing of potential peptide substrates without the constraints imposed by the phosphocellulose filter paper method (the need to incorporate positively charged residues into the peptide sequence). The technique combines the chelation of 32Pi by acid molybdate with PEI-cellulose chromatography. In this way the migration of 32P-labeled Pi, ATP, and protein are impeded while phosphopeptide is eluted in 1.5 ml from a 0.25-ml disposable column. In order to validate the assay we used two angiotensin II analogues as peptide substrates for the protein tyrosine kinase pp60c-src. The assay results using the new procedure were compared to those of the phosphocellulose filter paper technique. We also demonstrated the use of this method to test linear and cyclic peptides that could not be assayed with the phosphocellulose paper technique. This assay will aid those who are attempting to determine the substrate specificity of protein kinases.     

Reply to ‘Measurement of nitrogenase activity in legume root nodules: In defense of the acetylene reduction assay’ by J.K. Vessey

This article is in response to that of Vessey (1994) who argues that the traditional, closed acetylene reduction assay can still be a valuable tool for measuring relative differences in nitrogenase activity of legumes. To counter this assertion we consider the practical uses of the traditional assay procedure in relation to real research situations. This requires the use of the assay to be considered separately in the different circumstances of pot-grown and field-grown plants. We conclude that for pot-grown legumes there are a few practical applications where the use of the traditional, closed assay procedure is valid and we accept that these can be extended by the careful use of calibrations against open, flow-through systems. However, we doubt that there are many situations where such a calibration approach would have practical advantages over using the flow-through system to obtain the actual measurements. We cannot recommend any form of the uncalibrated acetylene reduction assay for field-based studies and suggest that researchers consider the merits of simple, alternative measurements such as dry weight, yield and total nitrogen.     

Progesterone receptor assays in low-protein cytosols: a modified charcoal-gelatin procedure

Quantitative measurement of steroid receptors including progesterone receptor (PgR) is usually accomplished by the dextran-coated charcoal (DCC) assay. At protein concentrations below about 1 mg/ml, however, serious underestimation of receptor content by DCC occurs, presumably because of adsorption of receptor to the charcoal and possibly to assay tubes, etc. We have therefore developed a modified charcoal-gelatin (MCG) procedure which largely avoids receptor losses even in samples with extremely low protein concentrations. In this MCG procedure, 0.1% gelatin is added to both sample and charcoal suspension, the charcoal content is increased to 1%, and dextran is no longer necessary. Comparison of the MCG procedure with the standard DCC and several other methods at decreasing protein concentrations shows that MCG retains acceptable efficiency for PgR at much lower protein than the others, even as low as 10 micrograms/ml. This MCG procedure will be useful in determining receptors for prognosis in very small human breast cancer biopsies, as shown here, but also for receptor determination in very small tissues such as specific brain regions, and for receptor assay during purification.