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  • UFC901024 0000234282

    Document Type:
    Certificate of Quality
    Lot Number:
    0000234282
    Product Catalog Number:
    UFC901024
    Product Catalog Name:
    Amicon® Ultra-15 Centrifugal Filter Unit

  • Large-Scale Filter-Aided Sample Preparation Method for the Analysis of the Ubiquitinome. 28260372

    Protein ubiquitination regulates key cellular functions, including protein homeostasis and signal transduction. The digestion of ubiquitinated proteins with trypsin yields a glycine-glycine remnant bound to the modified lysine residue (K-ε-GG) that can be recognized by specific antibodies for immunoaffinity purification (IAP) and subsequent identification of ubiquitination sites by mass spectrometry. Previous ubiquitinome studies based on this strategy have consistently digested milligram amounts of protein as starting material using in-solution digestion protocols prior to K-ε-GG enrichment. Filter-aided sample preparation (FASP) surpasses in-solution protein digestion in cleavage efficiency, but its performance has thus far been shown for digestion of sample amounts on the order of micrograms. Because cleavage efficiency is pivotal in the generation of the K-ε-GG epitope recognized during IAP, here we developed a large-scale FASP method (LFASP) for digestion of milligram amounts of protein and evaluated its applicability to the study of the ubiquitinome. Our results demonstrate that LFASP-based tryptic digestion is efficient, robust, reproducible, and applicable to the study of the ubiquitinome. We benchmark our results with state-of-the-art ubiquitinome studies and show a ∼3-fold reduction in the proportion of miscleaved peptides with the method presented here. Beyond ubiquitinome analysis, LFASP overcomes the general limitation in sample capacity of standard FASP-based protocols and can therefore be used for a variety of applications that demand a large(r) amount of starting material.
    Document Type:
    Reference
    Product Catalog Number:
    UFC901024
    Product Catalog Name:
    Amicon® Ultra-15 Centrifugal Filter Unit

  • A complex of Protocadherin-19 and N-cadherin mediates a novel mechanism of cell adhesion. 22184198

    During embryonic morphogenesis, adhesion molecules are required for selective cell-cell interactions. The classical cadherins mediate homophilic calcium-dependent cell adhesion and are founding members of the large and diverse cadherin superfamily. The protocadherins are the largest subgroup within this superfamily, yet their participation in calcium-dependent cell adhesion is uncertain. In this paper, we demonstrate a novel mechanism of adhesion, mediated by a complex of Protocadherin-19 (Pcdh19) and N-cadherin (Ncad). Although Pcdh19 alone is only weakly adhesive, the Pcdh19-Ncad complex exhibited robust adhesion in bead aggregation assays, and Pcdh19 appeared to play the dominant role. Adhesion by the Pcdh19-Ncad complex was unaffected by mutations that disrupt Ncad homophilic binding but was inhibited by a mutation in Pcdh19. In addition, the complex exhibited homophilic specificity, as beads coated with Pcdh19-Ncad did not intermix with Ncad- or Pcdh17-Ncad-coated beads. We propose a model in which association of a protocadherin with Ncad acts as a switch, converting between distinct binding specificities.
    Document Type:
    Reference
    Product Catalog Number:
    UFC901024
    Product Catalog Name:
    Amicon® Ultra-15 Centrifugal Filter Unit

  • Cross-repressive interactions between Lrig3 and netrin 1 shape the architecture of the inner ear. 19004851

    The sense of balance depends on the intricate architecture of the inner ear, which contains three semicircular canals used to detect motion of the head in space. Changes in the shape of even one canal cause drastic behavioral deficits, highlighting the need to understand the cellular and molecular events that ensure perfect formation of this precise structure. During development, the canals are sculpted from pouches that grow out of a simple ball of epithelium, the otic vesicle. A key event is the fusion of two opposing epithelial walls in the center of each pouch, thereby creating a hollow canal. During the course of a gene trap mutagenesis screen to find new genes required for canal morphogenesis, we discovered that the Ig superfamily protein Lrig3 is necessary for lateral canal development. We show that this phenotype is due to ectopic expression of the axon guidance molecule netrin 1 (Ntn1), which regulates basal lamina integrity in the fusion plate. Through a series of genetic experiments, we show that mutually antagonistic interactions between Lrig3 and Ntn1 create complementary expression domains that define the future shape of the lateral canal. Remarkably, removal of one copy of Ntn1 from Lrig3 mutants rescues both the circling behavior and the canal malformation. Thus, the Lrig3/Ntn1 feedback loop dictates when and where basement membrane breakdown occurs during canal development, revealing a new mechanism of complex tissue morphogenesis.
    Document Type:
    Reference
    Product Catalog Number:
    AB2034
    Product Catalog Name:
    Anti-Laminin Antibody