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  • Entrapping of impermeant probes of different size into nonpermeabilized synaptosomes as a method to study presynaptic mechanisms. 10617148

    Small molecules present during brain tissue homogenization are known to be entrapped within subsequently isolated synaptosomes. We have revisited this technique in view of its systematic utilization to incorporate into nerve endings impermeant probes of large size. Rat neocortical synaptosomes were prepared in the absence or in the presence of each of the following compounds: 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), tetanus toxin (TeTx) or its light chain (TeTx-LC), pertussis toxin (PTx), anti-syntaxin, or anti-SNAP25 monoclonal antibodies. Release of endogenous GABA and glutamate was then evoked by high K+ depolarization. GABA and glutamate overflows were inhibited by entrapped BAPTA and in synaptosomes prepared by homogenization in the presence of varying concentrations of TeTx or TeTx-LC. When synaptobrevin cleavage in synaptosomes entrapped with TeTx was monitored by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by western blotting, the extent of proteolysis was found to correspond quantitatively to that of release inhibition. GABA and glutamate overflows were increased by entrapped PTx; moreover, (-)-baclofen inhibited amino acid overflow more potently in standard than in PTx-containing synaptosomes. The overflows of GABA and glutamate were similarly decreased following incorporation of anti-syntaxin or anti-SNAP25 antibodies. Synaptosomal entrapping may be routinely used to internalize membrane-impermeant agents of different size in studies of presynaptic mechanisms.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple

  • [Monoclonal antibodies--new probes for diagnosis and therapy. Their use as an example of the micrometastasizing of solid tumors] 3293575

    Monoclonal antibody CK2, recognizing component No. 18, appeared to be the most suitable reagent for the detection of epithelial tumor cells in bone marrow. Its specificity was confirmed in a double-marker staining procedure (combination of APAAP-technique and radioautography). CK2 positive cells were demonstrated not to reveal any cross-reactivity with an antibody directed against the "leucocyte common antigen". A significant correlation between the presence of epithelial tumor cells in bone marrow and certain conventional risk factors was found. A more detailed phenotypic characterisation could demonstrate the expression of proliferation associated antigens on these cells. Furthermore in an immunotherapeutic approach with monoclonal antibody 17-1A, labelling of the disseminated tumor cells in bone marrow after infusion of the antibody was shown.
    Document Type:
    Reference
    Product Catalog Number:
    MAB3404
    Product Catalog Name:
    Anti-Cytokeratin 18 Antibody, clone CK2

  • Phage-based molecular probes that discriminate force-induced structural states of fibronectin in vivo. 22529344

    Applied forces and the biophysical nature of the cellular microenvironment play a central role in determining cellular behavior. Specifically, forces due to cell contraction are transmitted into structural ECM proteins and these forces are presumed to activate integrin "switches." The mechanism of such switches is thought to be the partial unfolding of integrin-binding domains within fibronectin (Fn). However, integrin switches remain largely hypothetical due to a dearth of evidence for their existence, and relevance, in vivo. By using phage display in combination with the controlled deposition and extension of Fn fibers, we report the discovery of peptide-based molecular probes capable of selectively discriminating Fn fibers under different strain states. Importantly, we show that the probes are functional in both in vitro and ex vivo tissue contexts. The development of such tools represents a critical step in establishing the relevance of theoretical mechanotransduction events within the cellular microenvironment.
    Document Type:
    Reference
    Product Catalog Number:
    AB2040
    Product Catalog Name:
    Anti-Fibronectin Antibody

  • Custom-designed MLPA using multiple short synthetic probes: application to methylation analysis of five promoter CpG islands in tumor and urine specimens from patients wi ... 20413679

    Ligation of two oligonucleotide probes hybridized adjacently to a DNA template has been widely used for detection of genome alterations. The multiplex ligation-dependent probe amplification (MLPA) technique allows simultaneous screening of multiple target sequences in a single reaction by using pairs of probes that carry tails for binding of common amplification primers. Resolution of the various targets is achieved by electrophoresis on the basis of predefined differences in amplicon length. In the conventional MLPA approach, one of the two target probes is generated by cloning in a single-stranded bacteriophage vector to introduce a sequence of defined length between the primer binding site and the specific target sequence. Here we demonstrate that differences in amplicon length can be achieved by using multiple short synthetic probes for each target sequence. When joined by a DNA ligase, these probes will form a single amplifiable template whose length is defined by the number and lengths of the individual probes. We have used this principle to establish a methylation-specific MLPA (MS-MLPA) assay that simultaneously determines the methylation status of five promoter CpG islands, and we have used this assay to analyze DNA from tumor tissue and corresponding urine samples from patients with bladder cancer. Our data show that the use of multiple short synthetic probes provides a simple means for custom-designed MS-MLPA analysis.
    Document Type:
    Reference
    Product Catalog Number:
    2100
    Product Catalog Name:
    Protein-Concentrate Kit (Micro)

  • Optimization of activity-based probes for proteomic profiling of histone deacetylase complexes. 18217751

    Histone deacetylases (HDACs) are key enzymatic regulators of the epigenome and serve as promising targets for anticancer therapeutics. Recently, we developed a photoreactive clickable probe, SAHA-BPyne, to report on HDAC activity and complex formation in native biological systems. Here, we investigate the selectivity, sensitivity, and inhibitory properties of SAHA-BPyne and related potential activity-based probes for HDACs. While we identified several probes that are potent HDAC inhibitors and label HDAC complex components in native proteomic preparations, SAHA-BPyne was markedly superior for profiling HDAC activities in live cells. Interestingly, the enhanced performance of SAHA-BPyne as an in situ activity-based probe could not be solely ascribed to potency in HDAC binding, implying that other features of the molecule were key to efficient active site-directed labeling in living systems. Finally, we demonstrate the value of in situ profiling of HDACs by comparing the activity and expression of HDAC1 in cancer cells treated with the cytotoxic agent parthenolide. These results underscore the utility of activity-based protein profiling for studying HDAC function and may provide insight for the future development of click chemistry-based photoreactive probes for the in situ analysis of additional enzyme activities.
    Document Type:
    Reference
    Product Catalog Number:
    06-720
    Product Catalog Name:
    Anti-HDAC1 Antibody

  • Site-specific inhibitors of NADPH oxidase activity and structural probes of flavocytochrome b: characterization of six monoclonal antibodies to the p22phox subunit. 15585859

    The integral membrane protein flavocytochrome b (Cyt b) is the catalytic core of the human phagocyte NADPH oxidase, an enzyme complex that initiates a cascade of reactive oxygen species important in the elimination of infectious agents. This study reports the generation and characterization of six mAbs (NS1, NS2, NS5, CS6, CS8, and CS9) that recognize the p22(phox) subunit of the Cyt b heterodimer. Each of the mAbs specifically detected p22(phox) by Western blot analysis but did not react with intact neutrophils in FACS studies. Phage display mapping identified core epitope regions recognized by mAbs NS2, NS5, CS6, CS8, and CS9. Fluorescence resonance energy transfer experiments indicated that mAbs CS6 and CS8 efficiently compete with Cascade Blue-labeled mAb 44.1 (a previously characterized, p22(phox)-specific mAb) for binding to Cyt b, supporting phage display results suggesting that all three Abs recognize a common region of p22(phox). Energy transfer experiments also suggested the spatial proximity of the mAb CS9 and mAb NS1 binding sites to the mAb 44.1 epitope, while indicating a more distant proximity between the mAb NS5 and mAb 44.1 epitopes. Cell-free oxidase assays demonstrated the ability of mAb CS9 to markedly inhibit superoxide production in a concentration-dependent manner, with more moderate levels of inhibition observed for mAbs NS1, NS5, CS6, and CS8. A combination of computational predictions, available experimental data, and results obtained with the mAbs reported in this study was used to generate a novel topology model of p22(phox).
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple

  • Polyclonal and monoclonal antibodies as probes of rat hepatic cytochrome P-450 isozymes. 3297805

    Cytochrome P-450 is the terminal oxidase of an electron transport system that is responsible for the oxidative metabolism of a large variety of endogenous and exogenous compounds. This broad substrate selectivity is caused by multiple isozymes of cytochrome P-450 and the wide substrate selectivity of many of these isozymes. We have isolated 11 isozymes of cytochrome P-450 from the livers of rats (cytochromes P-450a-P-450k). We have found both polyclonal and monoclonal antibodies increasingly useful to distinguish among these isozymes and to quantitate enzyme levels in liver microsomal preparations where as many as 15 or more cytochrome P-450 isozymes are present. Several of these isozymes show considerable immunochemical relatedness to each other, and operationally they can be grouped into families of immunochemically related isozymes that include cytochromes P-450b and P-450e in one family, cytochromes P-450c and P-450d in another, and cytochromes P-450f-P-450i, and P-450k in a third family. Immunoquantitation of some of these isozymes has revealed dramatic increases of over 50-fold in the levels of certain of these isozymes when exogenous compounds are administered to rats.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple