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  • 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.
    Document Type:
    Reference
    Product Catalog Number:
    16-200
    Product Catalog Name:
    Anti-Histidine Tagged Antibody, clone 4D11, biotin conjugate

  • Comprehensive identification of phosphorylation sites in postsynaptic density preparations. 16452087

    In the mammalian central nervous system, the structure known as the postsynaptic density (PSD) is a dense complex of proteins whose function is to detect and respond to neurotransmitter released from presynaptic axon terminals. Regulation of protein phosphorylation in this molecular machinery is critical to the activity of its components, which include neurotransmitter receptors, kinases/phosphatases, scaffolding molecules, and proteins regulating cytoskeletal structure. To characterize the phosphorylation state of proteins in PSD samples, we combined strong cation exchange (SCX) chromatography with IMAC. Initially, tryptic peptides were separated by cation exchange and analyzed by reverse phase chromatography coupled to tandem mass spectrometry, which led to the identification of phosphopeptides in most SCX fractions. Because each of these individual fractions was too complex to characterize completely in single LC-MS/MS runs, we enriched for phosphopeptides by performing IMAC on each SCX fraction, yielding at least a 3-fold increase in identified phosphopeptides relative to either approach alone (SCX or IMAC). This enabled us to identify at least one site of phosphorylation on 23% (287 of 1,264) of all proteins found to be present in the postsynaptic density preparation. In total, we identified 998 unique phosphorylated peptides, mapping to 723 unique sites of phosphorylation. At least one exact site of phosphorylation was determined on 62% (621 of 998) of all phosphopeptides, and approximately 80% of identified phosphorylation sites are novel.
    Document Type:
    Reference
    Product Catalog Number:
    AB1504
    Product Catalog Name:
    Anti-Glutamate receptor 1 Antibody

  • Phosphorylation state of postsynaptic density proteins. 15748150

    The postsynaptic density (PSD) is an electron-dense structure located at the synaptic contacts between neurons. Its considerable complexity includes cytoskeletal and scaffold proteins, receptors, ion channels and signaling molecules, in line with the role of PSDs in signal transduction and processing. The phosphorylation state of components of the PSD is central to synaptic transmission and is known to play a role in synaptic plasticity, learning and memory. The presence of a range of kinases and phosphatases in the PSD defines potential key players in this context. However, the substrates that these enzymes target have not been fully identified to date. We analyzed the protein composition of purified PSD samples from adult mouse brains by strong cation exchange chromatography fractionation of a tryptic digest followed by nano-reverse phase liquid chromatography coupled with electrospray ionization-quadrupole time of flight tandem mass spectrometry. This led to the identification of 244 proteins. To gain an insight into the phosphoproteome of the PSD we then purified phosphorylated tryptic peptides by immobilized metal ion affinity chromatography. This approach for the specific enrichment of phosphopeptides resulted in the identification of 42 phosphoproteins in the PSD preparation, 39 of which are known PSD components. Here we present a total of 83 in vivo phosphorylation sites.
    Document Type:
    Reference
    Product Catalog Number:
    AB1506
    Product Catalog Name:
    Anti-Glutamate Receptor 2 & 3 Antibody

  • DETERMINIG IN VIVO PHOSPHORYLATION SITES USING MASS SPECTROMETRY 22470061

    Phosphorylation is the most studied protein post-translational modification (PTM) in biological systems, since it controls cell growth, proliferation, survival, and other processes. High-resolution/high mass accuracy mass spectrometers are used to identify protein phosphorylation sites due to their speed, sensitivity, selectivity, and throughput. The protocols described here focus on two common strategies: (1) identifying phosphorylation sites from individual proteins and small protein complexes, and (2) identifying global phosphorylation sites from whole-cell and tissue extracts. For the first, endogenous or epitope-tagged proteins are typically immunopurified from cell lysates, purified via gel electrophoresis or precipitation, and enzymatically digested into peptides. Samples can be optionally enriched for phosphopeptides using immobilized metal affinity chromatography (IMAC) or titanium dioxide (TiO(2)) and then analyzed by microcapillary liquid chromatography/tandem mass spectrometry (LC-MS/MS). Global phosphorylation site analyses that capture pSer/pThr/pTyr sites from biological sources sites are more resource and time consuming and involve digesting the whole-cell lysate, followed by peptide fractionation by strong cation-exchange chromatography, phosphopeptide enrichment by IMAC or TiO(2), and LC-MS/MS. Alternatively, the protein lysate can be fractionated by SDS-PAGE, followed by digestion, phosphopeptide enrichment, and LC-MS/MS. One can also immunoprecipitate only phosphotyrosine peptides using a pTyr antibody followed by LC-MS/MS.
    Document Type:
    Reference
    Product Catalog Number:
    C5737
    Product Catalog Name:
    ZipTip® Pipette Tips
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