β-Naphthyl Acetate in Acetone Produces a Dark Background for Staining of Esterase Activity on Gel.
Ontology highlight
ABSTRACT:
SUBMITTER: Kumar D
PROVIDER: S-EPMC6486925 | biostudies-literature |
REPOSITORIES: biostudies-literature
Similar Datasets
Project description:α-Naphthyl acetate esterase (α-NAE) and acid α-naphthyl acetate esterase (ANAE), a class of special esterases, are important for lymphocyte typing and immunocompetence-monitoring. As such, the simultaneous detection of α-NAE and ANAE has become a target to effectively improve the accuracy in lymphocyte typing. Therefore, we developed a dual-factor synergistically activated ESIPT-based probe (HBT-NA) to detect α-NAE and ANAE sensitively, rapidly, and simultaneously in a differential manner. HBT-NA exhibits differential fluorescence signal outputs toward small changes of α-NAE and ANAE activities. HBT-NA displays a weak fluorescence signal at 392 nm over a pH range from 6.0 to 7.4. However, when it interacts with α-NAE (0-25 U) at pH = 7.4, the fluorescence intensity at 392 nm enhanced linearly within 60 s (F392 nm/F0392 nm = 0.042 Cα-NAE + 1.1, R2 = 0.99). Furthermore, HBT-NA emits ratiometric fluorescence signals (F505 nm/F392 nm) for ANAE (0-25 U) at pH = 6.0 within 2.0 min, exhibiting a good linear relationship (F505 nm/F392 nm = 0.83CANAE - 1.75, R2 = 0.99). The differential fluorescence signals can be used to simultaneously detect the activities of α-NAE and ANAE in solutions and complex living organisms. More importantly, based on the differential fluorescence signals toward α-NAE and ANAE, T lymphocytes and B lymphocytes could be successfully typed and differentiated among nontyped lymphocytes, facilitating the real-time evaluation of their immune functions using flow cytometry. Hence, HBT-NA could be used for the ultrasensitive detection of the enzyme activities of α-NAE and ANAE, the real-time precise typing of lymphocytes, and the monitoring of immunocompetence.
| S-EPMC8728733 | biostudies-literature
Project description:A 4-year, 7-month-old Holstein cow presented with anorexia. Physical examination revealed masses in the interscapular region and vagina. Blast cells were detected in the masses and peripheral blood by fine needle aspiration cytology and hematological examination. By bone marrow aspiration, blast cells constituted up to 24.2% of all nucleated cells, and 22% and 2% of non-erythroid cells stained positive for myeloperoxidase and alpha-naphthyl acetate esterase (ANAE), respectively. Pathological examination revealed the mass lesions consisted of a proliferation of tumor cells, which were positive for monocytic markers (HLA-DR and Iba-1). The cow was diagnosed with acute myelomonocytic leukemia (AMML). Even when tumor cells are ANAE-negative, AMML cannot be completely ruled out and should be considered when diagnosing cattle with leukemia/lymphoma.
| S-EPMC8636865 | biostudies-literature
Project description:BackgroundThe shortage of food based feedstocks has been one of the stumbling blocks in industrial biomanufacturing. The acetone bioproduction from the traditional acetone-butanol-ethanol fermentation is limited by the non-specificity of products and competitive utilization of food-based substrates. Using genetically modified Escherichia coli to produce acetone as sole product from the cost-effective non-food based substrates showed great potential to overcome these problems.ResultsA novel acetone biosynthetic pathway were constructed based on genes from Clostridium acetobutylicum (thlA encoding for thiolase, adc encoding for acetoacetate decarboxylase, ctfAB encoding for coenzyme A transferase) and Escherichia coli MG1655 (atoB encoding acetyl-CoA acetyltransferase, atoDA encoding for acetyl-CoA: acetoacetyl-CoA transferase subunit ? and ?). Among these constructs, one recombinant MG1655 derivative containing the hybrid pathway consisting of thlA, atoDA, and adc, produced the highest level of acetone from acetate. Reducing the gluconeogenesis pathway had little effect on acetone production, while blocking the TCA cycle by knocking out the icdA gene enhanced the yield of acetone significantly. As a result, acetone concentration increased up to 113.18 mM in 24 h by the resting cell culture coupling with gas-stripping methods.ConclusionsAn engineered E. coli strain with optimized hybrid acetone biosynthetic pathway can utilize acetate as substrate efficiently to synthesize acetone without other non-gas byproducts. It provides a potential method for industrial biomanufacturing of acetone by engineered E. coli strains from non-food based substrate.
| S-EPMC6330746 | biostudies-literature
Project description:To explore why some oligonucleotides in denaturing polyacrylamide gel could not be silver-stained, 134 different oligonucleotides were analyzed using denaturing polyacrylamide gel electrophoresis stained with silver and asymmetric cyanine. As a result, we found that the sensitivity of oligos (dA), (dC), (dG) and (dT) to silver staining could be ranged as (dA) > (dG) > (dC) > (dT) from high to low. It was unexpected that oligo (dT) was hard to be silver-stained. Moreover, the silver staining of an oligonucleotide containing base T could be partially or completely inhibited by base T. The inhibition of silver staining by base T was a competitive inhibition which could be affected by the amounts of the argyrophil nucleobase and base T, the cis-distance between the argyrophil nucleobase and base T, and the gel concentration. The changes of the intensity of an oligonucleotide band caused by the changes of DNA base composition were diverse and interesting. The intensity of some oligonucleotide bands would significantly change when the changes of DNA base composition accumulated to a certain extent (usually ? 4 nt). The sensitivity of cyanine staining of ? 11-nt long oligonucleotides could be enhanced about 250-fold by fixing the gels with methanol fixing solution.
| S-EPMC4674134 | biostudies-literature
Project description:The objective of the present study was to analyze serum protein complexes and detect serum esterase activities using nongradient blue native polyacrylamide gel electrophoresis (BN-PAGE). For analysis of potential protein complexes, serum from rat was used. Results demonstrate that a total of 8 gel bands could be clearly distinguished after Coomassie blue staining, and serum albumin could be isolated nearly as a pure protein. Moreover, proteins in these bands were identified by electrospray mass spectrometry and low-energy collision induced dissociation (CID)-MS/MS peptide sequencing and the existence of serum dihydrolipoamide dehydrogenase (DLDH) was confirmed. For studies of in-gel detection of esterase activities, serum from rat, mouse, and human was used. In-gel staining of esterase activity was achieved by the use of either ?-naphthylacetate or ?-naphthylacetate in the presence of Fast blue BB salt. There were three bands exhibiting esterase activities in the serum of both rat and mouse. In contrast, there was only one band showing esterase activity staining in the human serum. When serum samples were treated with varying concentrations of urea, esterase activity staining was abolished for all the bands except the one containing esterase 1 (Es1) protein that is known to be a single polypeptide enzyme, indicating that majority of these esterases were protein complexes or multimeric proteins. We also identified the human serum esterase as butyrylcholinesterase following isolation and partial purification using ammonium sulfate fractioning and ion exchange column chromatographies. Where applicable, demonstrations of the gel-based method for measuring serum esterase activities under physiological or pathophysiological conditions were illustrated. Results of the present study demonstrate that nongradient BN-PAGE can serve as a feasible analytical tool for proteomic and enzymatic analysis of serum proteins.
| S-EPMC3034814 | biostudies-literature
Project description:The title compound, C(12)H(12)Cl(2)O(2), has a bicyclic skeleton containing cyclo-hexene and benzene fragments. The cyclo-hexene ring adopts a half-chair conformation with displacements of two atoms out of the least-squares plane of 0.311?(2) and -0.336?(2)?Å. The Cl atoms are trans-positioned.
| S-EPMC2959845 | biostudies-other
Project description:Esterases are a large family of enzymes with wide applications in the industry. However, all esterases originated from natural sources, limiting their use in harsh environments or newly- emerged reactions. In this study, we designed a new esterase to develop a new protocol to satisfy the needs for better biocatalysts. The ideal spatial conformation of the serine catalytic triad and the oxygen anion hole at the substrate-binding site was constructed by quantum mechanical calculation. The catalytic triad and oxygen anion holes were then embedded in the protein scaffold using the new enzyme protocol in Rosetta 3. The design results were subsequently evaluated, and optimized designs were used for expression and purification. The designed esterase had significant lytic activities towards p-nitrophenyl acetate, which was confirmed by point mutations. Thus, this study developed a new protocol to obtain novel enzymes that may be useful in unforgiving environments or novel reactions.
| S-EPMC7587395 | biostudies-literature
Project description:Protein phosphorylation is one of the most common post-translational modifications regulating many cellular processes. The phos-tag technology was combined with two-dimensional zymograms, which consisted of non-reducing IEF PAGE or NEPHGE in the first dimension and high resolution clear native electrophoresis (hrCNE) in the second dimension. The combination of these electrophoresis methods was mild enough to accomplish in-gel activity staining for Fe(III)-reductases by NADH/Fe(III)-citrate/ferrozine, 3,3'-Diaminobenzidine/H2O2 or TMB/H2O2 in the second dimension. The phos-tag zymograms can be used to investigate phosphorylation-dependent changes in enzyme activity. Phos-tag zymograms can be combined with further downstream analysis like mass spectrometry. Non-reducing IEF will resolve proteins with a pI of 3-10, whereas non-reducing NEPHGE finds application for alkaline proteins with a pI higher than eight. Advantages and disadvantages of these new methods will be discussed in detail.
| S-EPMC4396385 | biostudies-literature
Project description:Oligosaccharide fragments of glycosaminoglycans may be separated for rapid analysis by electrophoresis through a 10% polyacrylamide matrix. An extensive ladder-like set of bands is observed for partial testicular hyaluronidase digests of chondroitin 4- or 6-sulphate, and for dermatan sulphate. Co-electrophoresis of purified oligosaccharides has established that the major bands of these patterns represent fragments differing in chain length by one disaccharide unit, with the smallest fragments having the greatest mobility. Additional minor bands, representing heterogeneity in the repeating unit structure, are also observed. There are slight differences in the mobilities of oligosaccharides derived from the three major types of sulphated glycosaminoglycans. Alcian Blue is employed for visualization of the digest fragments. Sample loads of 5-10 micrograms per band appear optimum. The smallest oligosaccharide which may be stained by this method is the hexasaccharide. After consideration of this effect, a good correlation is found to exist between densitometric scans of the gel-electrophoretic patterns and gel-filtration chromatographic profiles based on uronic acid concentration.
| S-EPMC1144100 | biostudies-other
Project description:Labeling substrates or products are paramount in determining enzymatic kinetic parameters. Several options are available; many laboratories use either radioactive or fluorescent labeling because of their high sensitivity. However, those methods have their own drawbacks such as half-life decay, expensive and hazardous. Here, we propose a novel, simple, economical and fast alternative to substrate labeling for studying the kinetics of nucleic acids: post-migration gel staining with SYBR Gold. Cleavage rates similar to the ones reported in the literature for the I-R3 DNA-cleaving DNA enzyme in the presence of zinc chloride are an indication of the quality of the new method. Moreover, the activity of the hammerhead ribozyme was also monitored by our method to illustrate its versatility. This labeling-free method has several advantages such as its ease of use as well as cost effective and versatility with both non-structured and structured RNAs or DNAs.
| S-EPMC7053750 | biostudies-literature