Journal: Biochemistry

Article Title: Differential misfolding properties of glaucoma-associated olfactomedin domains from human and mouse

doi: 10.1021/acs.biochem.8b01309

Figure Lengend Snippet: Aggregates of MBP-MmOLF isolated from E. coli are ThT-positive, a hallmark of amyloid. (A) Aligned chromatograms from Superdex-75 purifications of wild-type and mutant MBP-MmOLF fusion proteins. (B) ThT fluorescence of MBP-MmOLF aggregates compared to selected monomeric MBP-OLFs and (C) corresponding SDS-PAGE analysis demonstrates that, for equal amounts of protein, the average ThT fluorescence is similar for human and mouse.

Article Snippet: 16 E. coli Rosetta Gami 2 cells transformed with myocilin plasmid were inoculated to OD 600 ~ 0.1 and grown in Superior Broth (US Biological) at 37 °C to OD 600 of 0.8 to 1.0, cooled to 18 °C, then induced with 0.5 mM isopropyl-β-D-thiogalactopyranoside and 1 mM CaCl 2 overnight.

Techniques: Isolation, Mutagenesis, Fluorescence, SDS Page

Journal: Biomolecules

Article Title: Complement Activation May Drive the Pathogenicity of Anti-α6 and Anti-β4 Integrin Antibodies In Vivo

doi: 10.3390/biom16030417

Figure Lengend Snippet: Generation and characterization of anti-α6 and anti-β4 integrin IgG. ( A ) Schematic representation of the murine α6β4 integrin heterodimer. ( B ) Recombinant fragments of murine α6 integrin (amino acids 217–590) and murine β4 integrin (amino acids 1485–1817) were expressed in E. coli and affinity-purified. SDS-PAGE gel with PageBlueTM staining shows α6 integrin (lane 1) and β4 integrin (lane 4) fragments at approximately 120 kDa and 200 kDa, respectively. Molecular weight markers are shown in lanes 2 and 3. ( C ) Immunoblot analysis using sera from rabbits immunized with α6 or β4 integrin fragments detected corresponding bands in murine C5N keratinocyte lysates at approximately 120 kDa (lane 3) and 200 kDa (lane 8), respectively. Affinity-purified total IgG (lanes 4 and 9) and specific IgG (lanes 5 and 10) showed bands with the same molecular weights. Affinity-purified anti-α6 (lane 1) and anti-β4 integrin IgG (lane 6) showed similar results, whereas pre-immune rabbit sera (lanes 2 and 7) and normal rabbit (NR) IgG (lane 11) showed no detectable signals. The molecular weight marker is indicated on the right. ( D , E ) Indirect immunofluorescence microscopy confirmed binding of ( D ) anti-α6 integrin IgG and ( E ) anti-β4 integrin IgG to the epidermal side of the salt-split murine skin. DAPI (blue) was used to stain the nuclei. Scale bar: 50 µm.

Article Snippet: The corresponding murine fragments were expressed in E. coli Rosetta (DE3) cells (Novogene, Martinsried, Germany) as His-tag fusion proteins and purified using immobilized metal affinity chromatography.

Techniques: Recombinant, Affinity Purification, SDS Page, Staining, Molecular Weight, Western Blot, Marker, Immunofluorescence, Microscopy, Binding Assay