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  • Synaptoneurosome micromethod for fractionation of mouse and human brain, and primary neuronal cultures. 23017979

    Brain and primary neuron fractions enriched in synaptic terminals are important tools for neuroscientists in biochemical, neuroanatomical and physiological studies. We describe an annotated updated micro-method for preparing synaptoneurosomes (SNs) enriched in presynaptic and postsynaptic elements. An easy to follow, step-by-step, protocol is provided for making SNs from small amounts of mammalian brain tissue. This includes novel applications for material obtained from human neurosurgical procedures and primary rat neuronal cultures. Our updated method for preparing SNs using smaller amounts of tissue provides a valuable new tool and expands the capabilities of neuroscientists.
    Tipo de documento:
    Referencia
    Referencia del producto:
    03-100
    Nombre del producto:
    RIPAb+™ hnRNP M1-M4

  • Small molecules greatly improve conversion of human-induced pluripotent stem cells to the neuronal lineage. 22567022

    Efficient in vitro differentiation into specific cell types is more important than ever after the breakthrough in nuclear reprogramming of somatic cells and its potential for disease modeling and drug screening. Key success factors for neuronal differentiation are the yield of desired neuronal marker expression, reproducibility, length, and cost. Three main neuronal differentiation approaches are stromal-induced neuronal differentiation, embryoid body (EB) differentiation, and direct neuronal differentiation. Here, we describe our neurodifferentiation protocol using small molecules that very efficiently promote neural induction in a 5-stage EB protocol from six induced pluripotent stem cells (iPSC) lines from patients with Parkinson's disease and controls. This protocol generates neural precursors using Dorsomorphin and SB431542 and further maturation into dopaminergic neurons by replacing sonic hedgehog with purmorphamine or smoothened agonist. The advantage of this approach is that all patient-specific iPSC lines tested in this study were successfully and consistently coaxed into the neural lineage.
    Tipo de documento:
    Referencia
    Referencia del producto:
    Múltiplo
    Nombre del producto:
    Múltiplo

  • Cation exchange chromatography in antibody purification: pH screening for optimised binding and HCP removal. 17113367

    The production of pharmaceutical antibodies requires reliable and rapid processes with high purity and yield. Although protein A gels selectively and efficiently bind antibodies in the capture step, intense research is going on to find alternatives that can abolish the drawbacks of protein A chromatography. Ion exchangers e.g. are more robust, considerably cheaper and can eliminate ligand leaching. For the strong cation exchangers Fractogel EMD SO3- (M) and Fractogel EMD SE Hicap (M) we have evaluated the influence of pH for optimised binding and removal of host cell protein (HCP). In a fast initial screening we measured batch binding capacities. Subsequent scale-down to 96-well plate format proved that assay miniaturisation still provided reliable data. We demonstrated with the principle of residence time that scout columns are suitable for dynamic studies. The optimum pH range from batch binding was transferred to scout columns which were then used to screen for maximum dynamic capacities. In addition IEF titration curve analysis was employed to define a final operational pH. With this pH we ran labscale columns to purify monoclonal antibody. The cation exchangers showed high step yields and host cell proteins in the pools from gradient elution were reduced very effectively.
    Tipo de documento:
    Referencia
    Referencia del producto:
    16-200
    Nombre del producto:
    Anti-Histidine Tagged Antibody, clone 4D11, biotin conjugate

  • Spatial distribution of neural activity in the anterior olfactory nucleus evoked by odor and electrical stimulation. 21165975

    Several lines of evidence indicate that complex odorant stimuli are parsed into separate data streams in the glomeruli of the olfactory bulb, yielding a combinatorial "odotopic map." However, this pattern does not appear to be maintained in the piriform cortex, where stimuli appear to be coded in a distributed fashion. The anterior olfactory nucleus (AON) is intermediate and reciprocally interconnected between these two structures, and also provides a route for the interhemispheric transfer of olfactory information. The present study examined potential coding strategies used by the AON. Rats were exposed to either caproic acid, butyric acid, limonene, or purified air and the spatial distribution of Fos-immunolabeled cells was quantified. The two major subregions of the AON exhibited different results. Distinct odor-specific spatial patterns of activity were observed in pars externa, suggesting that it employs a topographic strategy for odor representation similar to the olfactory bulb. A spatially distributed pattern that did not appear to depend on odor identity was observed in pars principalis, suggesting that it employs a distributed representation of odors more similar to that seen in the piriform cortex. J. Comp. Neurol. 519:277-289, 2011. © 2010 Wiley-Liss, Inc.
    Tipo de documento:
    Referencia
    Referencia del producto:
    MAB5406
    Nombre del producto:
    Anti-GAD67 Antibody, clone 1G10.2

  • Tandem orthogonal proteolysis-activity-based protein profiling (TOP-ABPP)--a general method for mapping sites of probe modification in proteomes. 17545978

    Activity-based protein profiling (ABPP) utilizes active site-directed chemical probes to monitor the functional state of enzymes directly in native biological systems. Identification of the specific sites of probe labeling on enzymes remains a major challenge in ABPP experiments. In this protocol, we describe an advanced ABPP platform that utilizes a tandem orthogonal proteolysis (TOP) strategy coupled with mass spectrometric analysis to simultaneously identify probe-labeled proteins together with their exact sites of probe modification. Elucidation of probe modification sites reveals fundamental insights into the molecular basis of specific probe-protein interactions. The TOP-ABPP method can be applied to any type of proteomic sample, including those derived from in vitro or in vivo labeling experiments, and is compatible with a variety of chemical probe structures. Completion of the entire protocol, including chemical synthesis of key reagents, requires approximately 8-10 days.
    Tipo de documento:
    Referencia
    Referencia del producto:
    04-1037

  • Tgfbeta signal inhibition cooperates in the induction of iPSCs and replaces Sox2 and cMyc. 19765992

    Ectopic expression of Oct4, Sox2, cMyc, and Klf4 confers a pluripotent state upon several differentiated cell types, generating induced pluripotent stem cells (iPSCs) [1-8]. iPSC derivation is highly inefficient, and the underlying mechanisms are largely unknown. This low efficiency suggests the existence of additional cooperative factors whose identification is critical for understanding reprogramming. In addition, the therapeutic use of iPSCs relies on the development of efficient nongenetic means of factor delivery, and although a handful of replacement molecules have been identified, their use yields a further reduction to the already low reprogramming efficiency [9-11]. Thus, the identification of compounds that enhance rather than solely replace the function of the reprogramming factors will be of great use. Here, we demonstrate that inhibition of Tgfbbeta signaling cooperates in the reprogramming of murine fibroblasts by enabling faster, more efficient induction of iPSCs, whereas activation of Tgfbeta signaling blocks reprogramming. In addition to exhibiting a strong cooperative effect, the Tgfbeta receptor inhibitor bypasses the requirement for exogenous cMyc or Sox2, highlighting its dual role as a cooperative and replacement factor. The identification of a highly characterized pathway operating in iPSC induction will open new avenues for mechanistic dissection of the reprogramming process.
    Tipo de documento:
    Referencia
    Referencia del producto:
    AB5603
    Nombre del producto:
    Anti-Sox2 Antibody

  • The podosome marker protein Tks5 regulates macrophage invasive behavior. 22021214

    Tks5 is a Src substrate and adaptor protein previously recognized for its regulation of cancer cell invasion through modulation of specialized adhesion structures called podosomes/invadopodia. Here we show for the first time that Tks5 localizes to the podosomes of primary macrophages, and that Tks5 protein levels increase concurrently with podosome deposition during the differentiation of monocytes into macrophages. Similar results are reported for model THP-1 cells, which differentiate into macrophages and form proteolytically active podosomes in response to a PKC signaling agonist (PMA) and with sensitivity to a PKC inhibitor (bisindolylmaleimide). Genetic manipulation of Tks5 expression (silencing and overexpression) in stable THP-1 cell lines does not independently alter this macrophage differentiation process. Nor do these cells lose the ability to focalize F-actin and its accessory proteins into podosome-like structures following PMA treatment. However, Tks5 directly controls podosome-associated gelatin degradation and invasion through collective changes in adhesion, chemotaxis, and the expression/proteolytic activity of MMP9. The Src family kinase-dependent phosphorylation of Tks5 is also implicated in the regulation of THP-1 macrophage invasive behavior. These results therefore define a previously unappreciated function of Tks5 signaling specific to the functional attributes of the macrophage podosome in adhesion, motility, and extracellular matrix-remodeling.
    Tipo de documento:
    Referencia
    Referencia del producto:
    AB6004
    Nombre del producto:
    Anti-MT1-MMP Antibody, hinge region

  • APC16 is a conserved subunit of the anaphase-promoting complex/cyclosome. 20392738

    Error-free chromosome segregation depends on timely activation of the multi-subunit E3 ubiquitin ligase APC/C. Activation of the APC/C initiates chromosome segregation and mitotic exit by targeting critical cell-cycle regulators for destruction. The APC/C is the principle target of the mitotic checkpoint, which prevents segregation while chromosomes are unattached to spindle microtubules. We now report the identification and characterization of APC16, a conserved subunit of the APC/C. APC16 was found in association with tandem-affinity-purified mitotic checkpoint complex protein complexes. APC16 is a bona fide subunit of human APC/C: it is present in APC/C complexes throughout the cell cycle, the phenotype of APC16-depleted cells copies depletion of other APC/C subunits, and APC16 is important for APC/C activity towards mitotic substrates. APC16 sequence homologues can be identified in metazoans, but not fungi, by four conserved primary sequence stretches. We provide evidence that the C. elegans gene K10D2.4 and the D. rerio gene zgc:110659 are functional equivalents of human APC16. Our findings show that APC/C is composed of previously undescribed subunits, and raise the question of why metazoan APC/C is molecularly different from unicellular APC/C.
    Tipo de documento:
    Referencia
    Referencia del producto:
    3306

  • Miniature release events of glutamate from hippocampal neurons are influenced by the dystonia-associated protein torsinA. 22588999

    TorsinA is an evolutionarily conserved AAA+ ATPase, and human patients with an in-frame deletion of a single glutamate (ΔE) codon from the encoding gene suffer from autosomal-dominant, early-onset generalized DYT1 dystonia. Although only 30-40% of carriers of the mutation show overt motor symptoms, most experience enhanced excitability of the central nervous system. The cellular mechanism responsible for this change in excitability is not well understood. Here we show the effects of the ΔE-torsinA mutation on miniature neurotransmitter release from neurons. Neurotransmitter release was characterized in cultured hippocampal neurons obtained from wild-type, heterozygous, and homozygous ΔE-torsinA knock-in mice using two approaches. In the first approach, patch-clamp electrophysiology was used to record glutamate-mediated miniature excitatory postsynaptic currents (mEPSCs) in the presence of the Na(+) channel blocker tetrodotoxin (TTX) and absence of GABA(A) receptor antagonists. The intervals between mEPSC events were significantly shorter in neurons obtained from the mutant mice than in those obtained from wild-type mice. In the second approach, the miniature exocytosis of synaptic vesicles was detected by imaging the unstimulated release of FM dye from the nerve terminals in the presence of TTX. Cumulative FM dye release was higher in neurons obtained from the mutant mice than in those obtained from wild-type mice. The number of glutamatergic nerve terminals was also assessed, and we found that this number was unchanged in heterozygous relative to wild-type neurons, but slightly increased in homozygous neurons. Notably, in both heterozygous and homozygous neurons, the unitary synaptic charge during each mEPSC event was unchanged. Overall, our results suggest more frequent miniature glutamate release in neurons with ΔE-torsinA mutations. This change may be one of the underlying mechanisms by which the excitability of the central nervous system is enhanced in the context of DYT1 dystonia. Moreover, qualitative differences between heterozygous and homozygous neurons with respect to certain synaptic properties indicate that the abnormalities observed in homozygotes may reflect more than a simple gene dosage effect. Synapse 66:807-822, 2012. © 2012 Wiley Periodicals, Inc.
    Tipo de documento:
    Referencia
    Referencia del producto:
    AB5622
    Nombre del producto:
    Anti-Microtubule-Associated Protein 2 (MAP2) Antibody

  • Effects of a novel arginine methyltransferase inhibitor on T-helper cell cytokine production. 20345902

    The protein arginine methyltransferase (PRMT) family of enzymes catalyzes the transfer of methyl groups from S-adenosylmethionine to the guanidino nitrogen atom of peptidylarginine to form monomethylarginine or dimethylarginine. We created several less polar analogs of the specific PRMT inhibitor arginine methylation inhibitor-1, and one such compound was found to have improved PRMT inhibitory activity over the parent molecule. The newly identified PRMT inhibitor modulated T-helper-cell function and thus may serve as a lead for further inhibitors useful for the treatment of immune-mediated disease.
    Tipo de documento:
    Referencia
    Referencia del producto:
    Múltiplo
    Nombre del producto:
    Múltiplo