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  • 1.
    Eberle, Andrea B.
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics. Stockholm University, Faculty of Science, The Wenner-Gren Institute .
    Böhm, Stefanie
    Stockholm University, Faculty of Science, The Wenner-Gren Institute .
    Farrants, Ann-Kristin Östlund
    Stockholm University, Faculty of Science, The Wenner-Gren Institute .
    Visa, Neus
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    The use of a synthetic DNA-antibody complex as external reference for chromatin immunoprecipitation2012In: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 426, no 2, p. 147-152Article in journal (Refereed)
    Abstract [en]

    Chromatin immunoprecipitation (ChIP) is an analytical method used to investigate the interactions between proteins and DNA in vivo. ChIP is often used as a quantitative tool, and proper quantification relies on the use of adequate references for data normalization. However, many ChIP experiments involve analyses of samples that have been submitted to experimental treatments with unknown effects, and this precludes the choice of suitable internal references. We have developed a normalization method based on the use of a synthetic DNA-antibody complex that can be used as an external reference instead. A fixed amount of this synthetic DNA-antibody complex is spiked into the chromatin extract at the beginning of the ChIP experiment. The DNA-antibody complex is isolated together with the sample of interest, and the amounts of synthetic DNA recovered in each tube are measured at the end of the process. The yield of synthetic DNA recovery in each sample is then used to normalize the results obtained with the antibodies of interest. Using this approach, we could compensate for losses of material, reduce the variability between ChIP replicates, and increase the accuracy and statistical resolution of the data.

  • 2. Fedulova, Natalia
    et al.
    Mannervik, Bengt
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Experimental conditions affecting functional comparison of highly active glutathione transferases2011In: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 413, no 1, p. 16-23Article in journal (Refereed)
    Abstract [en]

    Glutathione transferases (GSTs, EC 2.5.1.18) possess multiple functions and have potential applications in biotechnology. Direct evidence of underestimation of activity of human GST A3-3 and porcine GST A2-2 measured at submicromolar enzyme concentrations is reported here for the first time. The combination of time-dependent and enzyme concentration-dependent loss of activity and the choice of the organic solvent for substrates were found to cause irreproducibility of activity measurements of GSTs. These effects contribute to high variability of activity values of porcine GST A2-2 and human Alpha-class GSTs reported in the literature. Adsorption of GSTs to surfaces was found to be the main explanation of the observed phenomena. Several approaches to improved functional comparison of highly active GSTs are proposed.

  • 3.
    Helmfors, Henrik
    et al.
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Eriksson, Jonas
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Langel, Ülo
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Optimized luciferase assay for cell-penetrating peptide-mediated delivery of short oligonucleotides2015In: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 484, p. 136-142Article in journal (Refereed)
    Abstract [en]

    An improved assay for screening for the intracellular delivery efficacy of short oligonucleotides using cell-penetrating peptides is suggested. This assay is an improvement over previous assays that use luciferase reporters for cell-penetrating peptides because it has been scaled up from a 24-well format to a 96-well format and no longer relies on a luciferin reagent that has been commercially sourced. In addition, the homemade luciferin reagent is useful in multiple cell lines and in different assays that rely on altering the expression of luciferase. To establish a new protocol, the composition of the luciferin reagent was optimized for both signal strength and longevity by multiple two-factorial experiments varying the concentrations of adenosine triphosphate, luciferin, coenzyme A, and dithiothreitol. In addition, the optimal conditions with respect to cell number and time of transfection for both short interfering RNA (siRNA) and splice-correcting oligonucleotides (SCOs) are established. Optimal transfection of siRNA and SCOs was achieved using the reverse transfection method where the oligonucleotide complexes are already present in the wells before the cells are plated. Z' scores were 0.73 for the siRNA assay and 0.71 for the SCO assay, indicating that both assays are suitable for high-throughput screening.

  • 4. Kjellander, Marcus
    et al.
    Mazari, Aslam M. A.
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Boman, Mats
    Mannervik, Bengt
    Stockholm University, Faculty of Science, Department of Neurochemistry. Uppsala University, Sweden.
    Johansson, Gunnar
    Glutathione transferases immobilized on nanoporous alumina: Flow system kinetics, screening, and stability2014In: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 446, p. 59-63Article in journal (Refereed)
    Abstract [en]

    The previously uncharacterized Drosophila melanogaster Epsilon-class glutathione transferases E6 and E7 were immobilized on nanoporous alumina. The nanoporous anodized alumina membranes were derivatized with 3-aminopropyl-triethoxysilane, and the amino groups were activated with carbonyldiimidazole to allow coupling of the enzymes via c-amino groups. Kinetic analyses of the immobilized enzymes were carried out in a circulating flow system using CDNB (1-chloro-2,4-dinitrobenzene) as substrate, followed by specificity screening with alternative substrates. A good correlation was observed between the substrate screening data for immobilized enzyme and corresponding data for the enzyme in solution. A limited kinetic study was also carried out on immobilized human GST S1-1 (also known as hematopoietic prostaglandin D synthase). The stability of the immobilized enzymes was virtually identical to that of enzymes in solution, and no leakage of enzyme from the matrix could be observed.

  • 5. Larsson, Emilia
    et al.
    Mannervik, Bengt
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Raffalli-Mathieu, Françoise
    Quantitative and selective polymerase chain reaction analysis of highly similar human alpha-class glutathione transferases2011In: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 412, no 1, p. 96-101Article in journal (Refereed)
    Abstract [en]

    Alpha-class glutathione transferases (GSTs) found expressed in human tissues constitute a family of four homologous enzymes with contrasting enzyme activities. In particular, GST A3-3 has been shown to contribute to the biosynthesis of steroid hormones in human cells and is selectively expressed in steroidogenic tissues. The more ubiquitous GST A1-1, GST A2-2, and GST A4-4 appear to be primarily involved in detoxification processes and are expressed at higher levels than GST A3-3. We are interested in studying the cell and tissue expression of the GST A3-3 gene, yet the existence of highly expressed sequence-similar homologs and of several splice variants is a serious challenge for the specific detection of unique transcript species. We found that published polymerase chain reaction (PCR) primers for GST A3-3 lack the specificity required for reliable quantitative analysis. Therefore, we designed quantitative PCR (qPCR) primers with greatly increased discrimination power for the human GSTA3 full-length transcript. The improved primers allow accurate discrimination between GST A3-3 and the other alpha-class GSTs and so are of great value to studies of the expression of the GSTA3 gene. The novel primers were used to quantify GSTA3 transcripts in human embryonic liver and steroidogenic cell lines.

  • 6.
    Saar, Külliki
    et al.
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Lindgren, Maria
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Hansen, Mats
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Eiríksdóttir, Emelía
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Jiang, Yang
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Rosenthal-Aizman, Katri
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Sassian, Meeri
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Langel, Ülo
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Cell-Penetrating Peptides: A Comparative Membrane Toxicity Study2005In: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 345, no 1, p. 55-65Article in journal (Refereed)
    Abstract [en]

    Cell-penetrating peptides (CPPs) constitute a new class of delivery vectors with high pharmaceutical potential. However, the abilities of these peptides to translocate through cell membranes can be accompanied by toxic effects resulting from membrane perturbation at higher peptide concentrations. Therefore, we investigated membrane toxicity of five peptides with well-documented cell-penetrating properties, pAntp(43–58), pTAT(48–60), pVEC(615–632), model amphipathic peptide (MAP), and transportan 10, on two human cancer cell lines, K562 (erythroleukemia) and MDA-MB-231 (breast cancer), as well as on immortalized aortic endothelial cells. We studied the effects of these five peptides on the leakage of lactate dehydrogenase and on the fluorescence of plasma membrane potentiometric dye bis-oxonol. In all cell lines, pAntp(43–58), pTAT(48–60), and pVEC(615–632) induced either no leakage or low leakage of lactate dehydrogenase, accompanied by modest changes in bis-oxonol fluorescence. MAP and transportan 10 caused significant leakage; in K562 and MDA-MB-231 cells, 40% of total lactate dehydrogenase leaked out during 10 min exposure to 10 μM of transportan 10 and MAP, accompanied by a significant increase in bis-oxonol fluorescence. However, none of the CPPs tested had a hemolytic effect on bovine erythrocytes comparable to mastoparan 7. The toxicity profiles presented in the current study are of importance when selecting CPPs for different applications.

  • 7.
    Zamani, Leila
    et al.
    Stockholm University, Faculty of Science, Department of Analytical Chemistry.
    Andersson, Fredrik O.
    Edebrink, Per
    Yang, Yang
    Jacobsson, Sven P.
    Stockholm University, Faculty of Science, Department of Analytical Chemistry.
    Conformational studies of a monoclonal antibody, IgG1, by chemical oxidation:Structural analysis by ultrahigh-pressure LC–electrospray ionizationtime-of-flight MS and multivariate data analysis2008In: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 380, no 2, p. 155-163Article in journal (Refereed)
    Abstract [en]

    We describe the development of a method in which protein oxidation by H2O2 followed by ultrahighpressure liquid chromatography (UHPLC) coupled with electrospray ionization time-of-flight mass spectrometry (ESI-ToFMS) and multivariate analysis are used to detect alterations in conformational states of proteins. In the study reported here, an IgG1 monoclonal antibody in native and denatured conformational states was oxidized by treatment with hydrogen peroxide. Peptide fragments generated by tryptic digestion were then analyzed by UHPLC-ESI-ToFMS. After reducing noise and extracting peaks from the LC–MS data using MzExplorer, software developed in-house and based on Matlab, we were able to distinguish peptides arising from the native and denatured states of the oxidized protein by principal component analysis. Peptides containing residues, which are inclined to undergo oxidation, such as methionine, are founded to be particularly important in this approach. We believe that the methodology could facilitate attempts to characterize the conformational states of recombinant monoclonal antibodies and other proteins.

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