References of "Analytical Biochemistry"
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See detailA nondetergent sulfobetaine improves protein unfolding reversibility in microcalorimetric studies
D'Amico, Salvino ULg; Feller, Georges ULg

in Analytical Biochemistry (2009), 385(2), 389-91

A nondetergent sulfobetaine (NDSB) was found to improve unfolding reversibility of several proteins by inhibiting heat-induced aggregation. As a consequence, DeltaH(cal)/DeltaH(vH) ratios were also ... [more ▼]

A nondetergent sulfobetaine (NDSB) was found to improve unfolding reversibility of several proteins by inhibiting heat-induced aggregation. As a consequence, DeltaH(cal)/DeltaH(vH) ratios were also improved to values close to 1 for a two-state unfolding. NDSB is effective in a wide range of pH values and especially at acidic pH generally used to calculate DeltaC(p) values by the Kirchhoff relation. The sulfobetaine also allows recording protein refolding by protecting the heat-induced unfolded state against aggregation. [less ▲]

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See detailDevelopment of a procedure to simultaneously isolate RNA, DNA, and proteins from characterizing cells invading or cultured on chitosan scaffolds.
Tchemtchoua Tateu, Victor ULg; Atanasova, Ganka; Aqil, Abdelhafid ULg et al

in Analytical Biochemistry (2009), 393(1), 145-7

For many years, chitosan and its derivatives have been considered to be promising biomaterials for tissue engineering and repair. However, information regarding their biological effect on cell phenotype ... [more ▼]

For many years, chitosan and its derivatives have been considered to be promising biomaterials for tissue engineering and repair. However, information regarding their biological effect on cell phenotype is usually limited to evaluation of cell proliferation and survival, overlooking proteomic and transcriptomic analysis. This is largely related to the lack of efficient and quantitative procedures for protein and nucleic acid purification from cells cultured on, or inside, chitosan scaffold. Here we describe an ultracentrifugation procedure enabling the simultaneous and quantitative recovery of high quality RNA, DNA and proteins from cells growing in close contact of biomaterial matrices containing chitosan. [less ▲]

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See detailA method to assess the lysosomal residence of proteins in cultured cells.
Gasingirwa, Christine; Thirion; Costa, Chrisostome ULg et al

in Analytical Biochemistry (2008), 374(2008), 3140

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See detailA nondetergent sulfobetaine prevents protein aggregation in microcalorimetric studies
Collins, T.; D'Amico, Salvino ULg; Georlette, D. et al

in Analytical Biochemistry (2006), 352(2), 299-301

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See detailNonisotopic substrate for assaying both human zinc and NAD+-dependent histone deacetylases.
Heltweg, Birgit; Dequiedt, Franck ULg; Verdin, Eric et al

in Analytical Biochemistry (2003), 319(1), 42-8

Histone deacetylases (HDACs) are involved in the regulation of transcription and their inhibitors are a promising class of new anticancer drugs. We have previously reported Boc(Ac)Lys-AMC, also termed MAL ... [more ▼]

Histone deacetylases (HDACs) are involved in the regulation of transcription and their inhibitors are a promising class of new anticancer drugs. We have previously reported Boc(Ac)Lys-AMC, also termed MAL, as a fluorescent substrate for HDACs. Now we present a modification of MAL called Z-MAL that is characterized by an increased rate of conversion by histone deacetylases of classes I and II and the recently discovered sirtuins (histone deacetylases class III). MAL and Z-MAL are the first nonradioactive substrates for class III enzymes. The new substrate Z-MAL allows for shorter assay times in inhibitor screening and is applicable to diverse sources of deacetylase activity even with completely different catalytic mechanisms. Interestingly, MAL shows some relative preference toward class II, indicating that subtype selectivity in small-molecule HDAC substrates might be obtained. [less ▲]

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See detailA general method for the chemical synthesis of gamma-P-32-labeled or unlabeled nucleoside 5 '-triphosphates and thiamine triphosphate
Bettendorff, Lucien ULg; Nghiem, Hoang O; Wins, Pierre et al

in Analytical Biochemistry (2003), 322(2), 190-197

Several methods for the chemical synthesis of gamma-P-32-labeled and unlabeled nucleoside 5'-triphosphates and thiamine triphosphate (ThTP) have been described. They often proved unsatisfactory because of ... [more ▼]

Several methods for the chemical synthesis of gamma-P-32-labeled and unlabeled nucleoside 5'-triphosphates and thiamine triphosphate (ThTP) have been described. They often proved unsatisfactory because of low yield, requirement for anhydrous solvents, procedures involving several steps or insufficient specific radioactivity of the labeled triphosphate. In the method described here, all these drawbacks are avoided. The synthesis of [gamma-P-32]TbTP was carried out in one step, using 1,3-dicyclohexyl carbodiimide as condensing agent for thiamine diphosphate and phosphoric acid in a dimethyl sulfoxide/pyridine solvent mixture. Anhydrous solvents were not required and the yield reached 90%. After purification, [gamma-P-32]ThTP had a specific radioactivity of 11 Ci/mmol and was suitable for protein phosphorylation. The method can also be used for the synthesis Of [gamma-P-32]ATP of the desired specific radioactivity. It can easily be applied to the synthesis of unlabeled ThTP or ribo- and deoxyribonucleoside 5'-triphosphates. In the latter case, inexpensive 5'-monophosphate precursors can be used as reactants in a 20-fold excess of phosphoric acid. Deoxyribonucleoside 5'-triphosphates were obtained in 6 h with a yield of at least 70%. After purification, the nucleotides were found to be suitable substrates for Taq polymerase during polymerase chain reaction cycling. Our method can easily be scaled up for industrial synthesis of a variety of labeled and unlabeled triphosphoric derivatives from their mono- or diphosphate precursors. (C) 2003 Elsevier Inc. All rights reserved. [less ▲]

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See detailSimultaneous Determination of Deoxyribonucleoside in the Presence of Ribonucleoside Triphosphates in Human Carcinoma Cells by High-Performance Liquid Chromatography
Decoster, L-A; Chen, X; Lejeune, F et al

in Analytical Biochemistry (1999), 270

Simultaneous determination of ribonucleoside and deoxyribonucleoside triphosphates in cells by HPLC is an analytical challenge since the concentration of dNTP present in mammalian cells is several orders ... [more ▼]

Simultaneous determination of ribonucleoside and deoxyribonucleoside triphosphates in cells by HPLC is an analytical challenge since the concentration of dNTP present in mammalian cells is several orders of magnitude lower than the corresponding NTP. Hence, the quantitation of dNTP in cells is generally performed after selective oxidation or removal of the major NTP. The procedures reported so far are lengthy and cumbersome and do not enable the simultaneous determination of NTP. We report the development of a simple, direct HPLC method for the simultaneous determination of dNTP and NTP in colon carcinoma WiDr cell extracts using a stepwise gradient elution ion-pairingHPLCwith uv detection at 260 nm and with a minimal chemical manipulation of cells. Exponentially growing WiDr cells were harvested by centrifugation, rinsed with phosphate- buffered saline, and carefully counted. The pellets were suspended in a known volume of ice-cold water and deproteinized with an equal volume of 6% trichloroacetic acid. The acid cell extracts (corresponding to 2.53 106 cells/100 ml) were centrifuged at 13,000g for 10 min at 4°C. The resulting supernatants were stored at 280°C prior to analysis. Aliquots (100 ml) were neutralized with 4.3 ml saturated Na2CO3 solution prior the injection of 40 ml onto the HPLC column (injection speed 250 ml/min). Chromatographic separations were performed using two Symmetry C18 3.5-mm (2 3 3.9 3 150 mm) columns (Waters), connected in series equipped with a Sentry guard column (3.9 3 20 mm i.d.) filled with the same packing material. The HPLC columns were kept at 30°C. The mobile phase was delivered at a flow rate of 0.5 ml/min, with the following stepwise gradient elution program: % solvent A/solvent B, 100/0 at 0 min 3 100/0 at 1 min 3 36/64 at 5 min 3 31/69 at 90 min 3 31/69 at 105 min 3 0/100 at 106 min 3 0/100 at 120 min; 50/50 MeOH/solvent B from 121 to 130 min; 100% solvent A from 131 to 160 min. Solvent A contained 0.01 M KH2PO4, 0.01 M tetrabutylammonium chloride, and 0.25% MeOH and was adjusted to pH 7.0 (550 ml 10 N NaOH for 1 liter solvent A). Solvent B consisted of 0.1 MKH2PO4, 0.028Mtetrabutylammonium chloride, and 30% MeOH and was neutralized to pH 7.0 (1.4 ml 10 N NaOH for 1 liter solvent B). Even though dNTPs are minor components of cell extracts, satisfactory regression coefficients were obtained for their calibration curves (r2 > 0.99) established with the addition–calibration methods up to 120 pmol/40-ml injection. The applicability of the method was demonstrated by in vitro studies of the modulation of NTP and dNTP pools in WiDr colon carcinoma cell lines exposed to various pharmacological concentrations of cytostatic drugs (i.e., FMdC, IUdR, gemcitabine). In conclusion, this optimized, simplified, analytical method enables the simultaneous quantitation of NTP and dNTP and may represent a valuable tool for the detection of minute alterations of cellular dNTP/NTP pools induced by anticancer/ antiviral drugs and diseases. © 1999 Academic Press [less ▲]

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See detailDetermination of Thiamin and Its Phosphate Esters in Cultured Neurons and Astrocytes Using an Ion-Pair Reversed-Phase High-Performance Liquid Chromatographic Method
Bettendorff, Lucien ULg; Peeters, Maryline; Jouan, Caroline ULg et al

in Analytical Biochemistry (1991), 198(1), 52-59

A sensitive method, based on fluorescence detection, for the determination of thiamin derivatives after precolumn derivatization is described. The separation is achieved on a PRP-1 column using ion-pair ... [more ▼]

A sensitive method, based on fluorescence detection, for the determination of thiamin derivatives after precolumn derivatization is described. The separation is achieved on a PRP-1 column using ion-pair reversed-phase HPLC. This method is especially well adapted to the detection of thiamin triphosphate in complex mixtures such as tissue extracts. The detection limit for TTP is 50 fmol. The contents of thiamin derivatives were determined in primary cultures of rat cerebellar granule neurons and cerebral astrocytes. The amount of TTP is about five times higher in neurons than in astrocytes. Thus in rat brain TTP seems to be essentially associated with neurons and the intracellular concentration is estimated to be about 0.2 microM. Our results suggest the existence, in nerve cells, of specific regulatory mechanisms not related to the blood-brain barrier and responsible for the maintenance of thiamin homeostasis in brain. [less ▲]

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See detailA Colorimetric Assay for the Simultaneous Measurement of Plasminogen Activators and Plasminogen Activator Inhibitors in Serum-Free Conditioned Media from Cultured Cells
Leprince, Pierre ULg; Rogister, Bernard ULg; Moonen, Gustave ULg

in Analytical Biochemistry (1989), 177(2), 341-6

The coupled photometric assay for plasminogen activator reported by Coleman and Green (1981) Methods in Enzymology (Lorand, L., Ed.), Vol. 80, pp. 408-414, Academic Press, San Diego, CA) has been adapted ... [more ▼]

The coupled photometric assay for plasminogen activator reported by Coleman and Green (1981) Methods in Enzymology (Lorand, L., Ed.), Vol. 80, pp. 408-414, Academic Press, San Diego, CA) has been adapted for use with 96-well plates and an automatic microplates spectrophotometer. The assay allows the discrimination between tissue-type and urokinase-type plasminogen activators in cell culture-conditioned media. It provides a level of detection of these enzymes in the range 10(-17) to 10(-13) mol (determined using purified human plasminogen activators), uses no radioisotopes, and is faster and more economical than similar assays using specific peptide substrates for plasminogen activators. Levels of free plasminogen activator inhibitor activity can be simultaneously measured on the same samples by a simple adaptation of the assay. This method allows an easy treatment of the data by interfacing with a computer and should thus be useful when large numbers of samples are assayed. [less ▲]

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See detailPreparation of reproducible alkaline phosphatase-antibody conjugates for enzyme immunoassay using a heterobifunctional linking agent
Jeanson, Antoinette; Cloes, Jean-Michel; Bouchet, Mireille et al

in Analytical Biochemistry (1988), 172(2), 392-396

Conjugates of alkaline phosphatase (AP) and mouse monoclonal immunoglobulins G (IgG) were prepared by means of the heterobifunctional linker, N-succinimidyl 3-(2-pyridyldithio)-propionate. The efficiency ... [more ▼]

Conjugates of alkaline phosphatase (AP) and mouse monoclonal immunoglobulins G (IgG) were prepared by means of the heterobifunctional linker, N-succinimidyl 3-(2-pyridyldithio)-propionate. The efficiency of such conjugates can be improved by optimizing the degree of substitution of IgG and AP. We have determined conditions yielding better performing conjugates than those synthesized by methods described previously. Moreover, the results obtained with the technique presented here are quite reproducible with all four monoclonal antibodies tested. [less ▲]

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See detailSynthesis of [Gamma-32p]Thiamine Triphosphate
Grandfils, Christian ULg; Bettendorff, Lucien ULg; de Rycker, C. et al

in Analytical Biochemistry (1988), 169(2), 274-278

We developed a novel chemical synthesis of thiamine triphosphate which allows us to incorporate 32P in the gamma position. The reaction is based on the condensation of [32P]orthophosphoric acid and ... [more ▼]

We developed a novel chemical synthesis of thiamine triphosphate which allows us to incorporate 32P in the gamma position. The reaction is based on the condensation of [32P]orthophosphoric acid and thiamine diphosphate in the presence of ethyl chloroformate. After purification by two ion-exchange purification steps, the thiamine derivative has a specific radioactivity of 10 Ci/mmol. The average final yield synthesis is about 10%. [less ▲]

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See detailHigh-Yield Synthesis of a (3H)Ethylenediamine Ditetrodotoxin Derivative
Bontemps, José; Cantineau, Robert; Grandfils, Christian ULg et al

in Analytical Biochemistry (1984), 139

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See detailAssay for radiolabeled type IV collagen in the presence of other proteins using a specific collagenase.
Garbisa, S.; Tryggvason, K.; Foidart, Jean-Michel ULg et al

in Analytical Biochemistry (1980), 107(1), 187-92

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See detailEnzyme-linked immunoassay (ELISA) for connective tissue components.
Rennard, S. I.; Berg, R.; Martin, G. R. et al

in Analytical Biochemistry (1980), 104(1), 205-14

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