Analysis for metallothioneins using coupled techniques

Łobiński, Ryszard; Chassaigne, Hubert; Szpunar, Joanna

doi:10.1016/s0039-9140(97)00343-3

Cited by 120 publications

(82 citation statements)

References 44 publications

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“…Saturation techniques such as Cd(II) saturation method [25,26], Ag(I) [27,28], and Hg(II) saturation assays [29,30], followed by immunochemical [31][32][33][34][35][36], separation [21,22,24,[37][38][39], electromigration [40][41][42][43][44][45], and electrochemical techniques [46][47][48][49][50][51][52][53][54][55][56][57][58][59][60][61][62][63][64][65] are most frequently used for MT quantification in biological samples. Information about the expression and function of single MT isoforms can be found by mRNA analysis using RT-PCR or expression microarray [66][67][68][69].…”

Section: Introductionmentioning

confidence: 99%

Using of chicken antibodies for metallothionein detection in human blood serum and cadmium‐treated tumour cell lines after dot‐ and electroblotting

et al. 2009

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Metallothionein (MT) is a low-molecular mass protein playing an essential role in homeostasis of heavy metal ions. Its relation with formation and progression of a tumour disease is discussed in this article. Here, we propose a new methodological approach for visualization of MT on PVDF membranes after dot- and electroblotting by using a commercial mouse monoclonal antibody E9 and polyclonal chicken antibodies. The optimized procedure was as follows. We dotted 1 microL sample volume on PVDF membrane and let it to dry. Then, we blocked the membrane surface with 2% BSA in PBS for 30 min. After that, the membrane was incubated in chicken primary antibody (diluted 1:500), washed, and incubated in rabbit-anti-chicken secondary antibody conjugated with horseradish peroxidase. To visualize the interaction, we used 3-aminoethyl-9-carbazole. Under these conditions, we estimated detection limit as 3 pg of MT per 1 microL. The optimal approach was further utilized for detection of MT level in two human fibroblast cell lines and in blood serum obtained from children with medulloblastoma. The results were in good agreement with differential pulse voltammetry-Brdicka reaction.

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Section: Introductionmentioning

confidence: 99%

Using of chicken antibodies for metallothionein detection in human blood serum and cadmium‐treated tumour cell lines after dot‐ and electroblotting

et al. 2009

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“…In CE, MT subisoforms are separated according to their charge-to-mass ratios, unlike the hydrophobicity-based separations of LC. CE is especially attractive due to its versatility, rapidity, low sample and reactive consumption and high separation efficiency [11][12][13].…”

Section: Introductionmentioning

confidence: 99%

Modeling the migration behavior of rabbit liver apothioneins in capillary electrophoresis

et al. 2008

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Metallothioneins (MTs) are a group of low-molecular-mass proteins (6-7 kDa) characterized by their great affinity for heavy metal ions. At acidic pH, the bound metal ions are released from the amino acidic structure and MTs lead to apothioneins. In this study, a general equation is used to model the electrophoretic mobility of rabbit liver apothioneins as a function of the pH of the separation electrolyte. The ability of these relationships to explain the migration behavior of these relatively complex polyprotic proteins in the pH range between 2 and 6 has been investigated. Their relevant ionization constant values in the studied pH range were estimated and employed for molecular charge calculations. The classical semiempirical relationships between electrophoretic mobility and charge-to-mass ratio (me vs. q/Malpha) were tested for prediction of their electrophoretic separations. The accuracy of the separations predicted at acidic pH was confirmed by CE-ESI-MS.

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“…Based on previously described methods, [23][24][25][26] the following procedure was used to extract the cytosol from the selected tissues of exposed T. fluviatilis. About 0.1 g of hepatopancreas and gills from each specimen was homogenized using 10 mL 0.05 mol L -1 TRIS (tris(hydroxymethyl)aminomethane) (pH 7.0 and 4 o C) in 30 mL centrifuge tubes.…”

Section: Sample Preparationmentioning

confidence: 99%

Bioconcentration of Cd and Pb by the river crab Trichodactylus fluviatilis (crustacea: decapoda)

Franchi

Menegário

Brossi-Garcia

et al. 2011

J. Braz. Chem. Soc.

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Foi avaliada a bioconcentração de cádmio e chumbo pelo caranguejo fluvial Trichodactylus fluviatilis. Trinta animais foram expostos a 200 mg L -1 de cádmio e chumbo por 7, 14 e 21 dias. Após dissecação e digestão nitro-perclórica, os metais foram determinados nas brânquias, hepatopâncreas e músculo por espectrometria de emissão óptica com plasma acoplado indutivamente. O chumbo foi detectado apenas nas brânquias, sem diferenças significativas entre diferentes períodos de exposição. O cádmio foi encontrado em todos os tecidos após exposição. Diferenças significativas entre as concentrações de cádmio em animais expostos por diferentes períodos sugerem um processo de acumulação, com concentração estabilizada após 14 dias. Usando um procedimento de extração em fase sólida com Saccharomyces cerevisae e com o objetivo de avaliar a transferência e estocagem de cádmio nos tecidos, foi realizado o fracionamento de cádmio na forma livre (ou lábil) e cádmio-proteína (possivelmente metalotioneína) (Cd-P) em animais expostos a 200 mg L -1 de cádmio por 21 dias. A determinação de cádmio livre e Cd-P nos tecidos mostrou que nas brânquias o metal foi encontrado principalmente na forma livre, enquanto que no hepatopâncreas o metal foi encontrado principalmente ligado à proteína. Pode-se inferir que, absorvido através das brânquias, o cádmio foi transferido e estocado no hepatopâncreas dos animais.The bioconcentration of cadmium and lead by the freshwater crab Trichodactylus fluviatilis was evaluated. Thirty animals were exposed to 200 mg L -1 of cadmium and lead for 7, 14 and 21 days. Both metals were determined in gills, hepatopancreas and muscle after dissection and digestion by inductively coupled plasma optical emission spectrometry. Lead was detected only in gills, but without significant difference among different exposure periods. Cadmium was found in all tissues after exposure. Significant differences among cadmium concentrations in animals exposed for different periods suggest an accumulation process, with concentration stabilized after 14 days. Fractionation of free (or labile) cadmium and cadmium protein (possibly metallothionein) (Cd-P) in gills and hepatopancreas were carried out to assess the cadmium transference and storage in the tissues using a solid phase extraction procedure with Saccharomyces cerevisae. Fractionation of free (or labile) cadmium and cadmium protein (possibly metallothionein) (Cd-P) in animals exposed to 200 mg L -1 for 21 days in gills and hepatopancreas were carried to assess the cadmium transference and storage in the tissues using a solid phase extraction procedure with Saccharomyces cerevisae. In gills, cadmium was found mainly in the free form, while in hepatopancreas the metal was found mainly bound to the protein (Cd-P). It may be inferred that, absorbed through gills, cadmium was transferred and stored in the hepatopancreas.

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Analysis for metallothioneins using coupled techniques

Cited by 120 publications

References 44 publications

Using of chicken antibodies for metallothionein detection in human blood serum and cadmium‐treated tumour cell lines after dot‐ and electroblotting

Using of chicken antibodies for metallothionein detection in human blood serum and cadmium‐treated tumour cell lines after dot‐ and electroblotting

Modeling the migration behavior of rabbit liver apothioneins in capillary electrophoresis

Bioconcentration of Cd and Pb by the river crab Trichodactylus fluviatilis (crustacea: decapoda)

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