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  • Measuring synaptosomal associated protein-25 kDa in human cerebral spinal fluid. 9789208

    Changes in the quantity and distribution of neuronal proteins have been reported in psychiatric and neurological illnesses. The majority of this work has been performed in post-mortem samples and the results are difficult to apply to clinical care. The objective of this study is to develop a methodology that can identify trace amounts of brain proteins in cerebral spinal fluid (CSF). Human cerebral spinal fluid was processed to remove albumin and immunoglobulins. CSF samples were analyzed on Western blots using a monoclonal antibody against SNAP-25. These samples were compared to SNAP-25 immunoprecipitated from CSF, rat and human brain homogenates. The monoclonal antibody Mab 331 identified a single band of 25 kDa in all samples. These results demonstrate that the presynaptic protein SNAP-25 can be identified and measured in CSF.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple

  • Measuring cell-type specific differential methylation in human brain tissue. 24000956

    The behavior of epigenetic mechanisms in the brain is obscured by tissue heterogeneity and disease-related histological changes. Not accounting for these confounders leads to biased results. We develop a statistical methodology that estimates and adjusts for celltype composition by decomposing neuronal and non-neuronal differential signal. This method provides a conceptual framework for deconvolving heterogeneous epigenetic data from postmortem brain studies. We apply it to find cell-specific differentially methylated regions between prefrontal cortex and hippocampus. We demonstrate the utility of the method on both Infinium 450k and CHARM data.
    Document Type:
    Reference
    Product Catalog Number:
    MAB377
    Product Catalog Name:
    Anti-NeuN Antibody, clone A60

  • Measuring dynamic changes in histone modifications and nucleosome density during activated transcription in budding yeast. 22183585

    Chromatin immunoprecipitation is widely utilized to determine the in vivo binding of factors that regulate transcription. This procedure entails formaldehyde-mediated cross-linking of proteins and isolation of soluble chromatin followed by shearing. The fragmented chromatin is subjected to immunoprecipitation using antibodies against the protein of interest and the associated DNA is identified using quantitative PCR. Since histones are posttranslationally modified during transcription, this technique can be effectively used to determine the changes in histone modifications that occur during transcription. In this paper, we describe a detailed methodology to determine changes in histone modifications in budding yeast that takes into account reductions in nucleosome.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple

  • Measuring ligand-dependent and ligand-independent interactions between nuclear receptors and associated proteins using Bioluminescence Resonance Energy Transfer (BRET). 17016546

    Bioluminescent resonance energy transfer (BRET2) is a recently developed technology for the measurement of protein-protein interactions in a live, cell-based system. BRET2 is characterized by the efficient transfer of excited energy between a bioluminescent donor molecule (Renilla luciferase) and a fluorescent acceptor molecule (a mutant of Green Fluorescent Protein (GFP2). The BRET2 assay offers advantages over fluorescence resonance energy transfer (FRET) because it does not require an external light source thereby eliminating problems of photobleaching and autoflourescence. The absence of contamination by light results in low background that permits detection of very small changes in the BRET2 signal. BRET2 is dependent on the orientation and distance between two fusion proteins and therefore requires extensive preliminary standardization experiments to conclude a positive BRET2 signal independent of variations in protein titrations and arrangement in tertiary structures. Estrogen receptor (ER) signaling is modulated by steroid receptor coactivator 1 (SRC-1). To establish BRET2 in a ligand inducible system we used SRC-1 as the donor moiety and ER as the acceptor moiety. Expression and functionality of the fusion proteins were assessed by transient transfection in HEK-293 cells followed by Western blot analysis and measurement of ER-dependent reporter gene activity. These preliminary determinations are required prior to measuring nuclear receptor protein-protein interactions by BRET2. This article describes in detail the BRET2 methodology for measuring interaction between full-length ER and coregulator proteins in real-time, in an in vivo environment.
    Document Type:
    Reference
    Product Catalog Number:
    MAB4400

  • Measuring GSK3 expression and activity in cells. 19099245

    Glycogen synthase kinase (GSK)-3 is a key signalling intermediate in the action ofWnts. This protein kinase is ubiquitously expressed and has high inherent activity but is inhibited by activation of Wnt signalling or activation of growth factor receptor tyrosine kinases (e.g. insulin, nerve growth factor [NGF], platelet-derived growth factor [PDGF], etc.). The degree of inhibition of GSK3 in cells treated with such reagents is dependent on the cell type and the stimulus used. Therefore, the ability to accurately measure GSK3 activity in cells is an important aspect of GSK3 research. The activity of GSK3 is reduced by posttranslational modification (phosphorylation) and this can be measured by immunoblot with specific reagents (indirect), or by immunoprecipitation and assay (direct), as long as the modification is protected during these procedures. However, inhibition by phosphorylation is specific to cellular activation by growth factors and nutrients. Wnt inhibition of GSK3 does not involve phosphorylation of these residues on GSK3 and therefore it cannot be measured using this modification. Currently, the simplest way to assess Wnt inhibition of GSK3 is to monitor phosphorylation of specific GSK3 substrates in cells (e.g. beta-catenin). Alternatively, Wnt inhibition of GSK3 can be measured by partial purification of cellular GSK3 by ion exchange chromatography and assay of fractions or possibly by immunoprecipitation and assay. In this chapter, we demonstrate the use of the different approaches to measure GSK3 activity in SH-SY5Y cells, describe the best antibodies currently available, and discuss the potential drawbacks of each method.
    Document Type:
    Reference
    Product Catalog Number:
    05-643
    Product Catalog Name:
    Anti-phospho-GSK3β (Ser9) Antibody, clone 2D3

  • Considerations when measuring urinary albumin: precision, substances that may interfere, and conditions for sample storage. 1764788

    The measurement of small but abnormal amounts of albumin in urine is important in evaluating kidney disease in people with diabetes mellitus, hypertension, or possible adverse health effects from exposure to nephrotoxins. Routine laboratory methods for measuring albumin are not sensitive enough to measure the amounts that are significant in urine (less than 30 mg/L). In our laboratory we used three immunoassays for measuring urinary albumin: enzyme-linked immunosorbent assay (EIA), radioimmunoassay (RIA), and immunoturbidimetric assay (IT). We calculated the CVs of the three methods, investigated potential interfering substances at three times their normal concentrations, and stored urine under different conditions to find the best way to protect the sample until assay. The potential interferents we checked were transferrin, urea, beta 2-microglobulin, retinol-binding protein, creatinine, kappa and lambda light chains, IgG, hemoglobin, ketone, and glucose. The stability study involved two study temperatures (-20 and -70 degrees C) and four treatments (centrifuging or filtering, before or after storage). We found the following: the RIA had the lowest CV; the results from the interference study showed no interference from normal physiological concentrations of the substances investigated; storage at -70 degrees C regardless of the treatment should be adequate to prevent loss of albumin immunoreactivity.
    Document Type:
    Reference
    Product Catalog Number:
    20-176
    Product Catalog Name:
    100X GTPγS, 10mM

  • A protein cross-linking assay for measuring cell surface expression of glutamate receptor subunits in the rodent brain after in vivo treatments. 22470150

    Trafficking of neurotransmitter receptors between intracellular and cell surface compartments is important for regulating neurotransmission. We developed a method for determining if an in vivo treatment has altered receptor distribution in a particular region of rodent brain. After the treatment, brain slices are rapidly prepared from the region of interest. Then, cell surface-expressed proteins are covalently cross-linked using the membrane-impermeable, bifunctional cross-linker bis(sulfosuccinimidyl)suberate (BS(3)). This increases the apparent molecular weight of surface receptors, while intracellular receptors are not modified. Thus, surface and intracellular receptor pools can be separated and quantified using SDS-PAGE and immunoblotting. This method is particularly useful for analyzing AMPA receptor subunits, offering advantages in accuracy, efficiency, and cost compared to biotinylation. A disadvantage is that some antibodies no longer recognize their target protein after cross-linking. We have used this method to quantify changes in receptor distribution after acute and chronic exposure to psychomotor stimulants.
    Document Type:
    Reference
    Product Catalog Number:
    AB1506
    Product Catalog Name:
    Anti-Glutamate Receptor 2 & 3 Antibody

  • Optical sensors for measuring dynamic changes of cytosolic metabolite levels in yeast. 22036883

    Optical sensors allow dynamic quantification of metabolite levels with subcellular resolution. Here we describe protocols for analyzing cytosolic glucose levels in yeast using genetically encoded Förster resonance energy transfer (FRET) sensors. FRET glucose sensors with different glucose affinities (Kd) covering the low nano- to mid- millimolar range can be targeted genetically to the cytosol or to subcellular compartments. The sensors detect the glucose-induced conformational change in the bacterial periplasmic glucose/galactose binding protein MglB using FRET between two fluorescent protein variants. Measurements can be performed with a single sensor or multiple sensors in parallel. In one approach, cytosolic glucose accumulation is measured in yeast cultures in a 96-well plate using a fluorimeter. Upon excitation of the cyan fluorescent protein (CFP), emission intensities of CFP and YFP (yellow fluorescent protein) are captured before and after glucose addition. FRET sensors provide temporally resolved quantitative data of glucose for the compartment of interest. In a second approach, reversible changes of cytosolic free glucose are measured in individual yeast cells trapped in a microfluidic platform, allowing perfusion of different solutions while FRET changes are monitored in a microscope setup. By using the microplate fluorimeter protocol, 96 cultures can be measured in less than 1 h; analysis of single cells of a single genotype can be completed in <2 h. FRET-based analysis has been performed with glucose, maltose, ATP and zinc sensors, and it can easily be adapted for high-throughput screening using a wide spectrum of sensors.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple