Journal: Proteome Science

Article Title: Proteomic analysis of laser capture microscopy purified myotendinous junction regions from muscle sections

doi: 10.1186/1477-5956-12-25

Figure Lengend Snippet: Selection of proteins identified in the MTJ sample

Article Snippet: The primary antibodies used in this work were: goat anti human FHL1 (cat. AHP2070, AbD Serotec), mouse monoclonal anti-syntrophin (cat. SAB4200213, Sigma-Aldrich), mouse monoclonal anti-myomesin (cat. mMaC myomesin B4, DSHB), mouse monoclonal anti-titin (cat. 9 D10, DSHB), mouse monoclonal anti s-laminin (cat. C4, DSHB), mouse monoclonal anti filaminC RR90 (an IgA sub-type [ ]), mouse monoclonal anti-Annexin I (cat. EH17a, DSHB), mouse monoclonal anti-myotilin (RSO34, Novocastra), mouse monoclonal anti-desmin (cat. D76, DSHB), rabbit polyclonal anti-TACP (cat. QC18385, Sigma-Aldrich), mouse monoclonal anti-αB crystallin (cat. CPTC-CRYAB-3, DSHB), mouse monoclonal anti-tubulin (cat. E7, DSHB), mouse monoclonal anti-α actinin (cat EA53, Sigma-Aldrich), mouse monoclonal anti-α-sarcoglycan (cat. IVD3(1)A9, DSHB).

Techniques: Selection, Membrane

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Recombinant human IgA1 and IgA2 autoantibodies to type VII collagen induce subepidermal blistering ex vivo.

doi: 10.4049/jimmunol.1400160

Figure Lengend Snippet: FIGURE 1. Recombinant autoantibodies demonstrated the correct m.w. under reducing and nonreducing conditions, labeled the DEJ and bound to type VII collagen extracted from human dermis. (A) Recombinant autoantibodies were separated by 12% SDS-PAGE under reducing conditions and stained with Coomassie blue. The H chains of the recombinant proteins migrated at 50 (rIgG1), 60 (rIgA1), and 55 kDa (rIgA2), and the k L chains migrated at 25 kDa. The m.w. standard is indicated on the left. (B) Equal amounts of recombinant autoantibodies were separated by 7% SDS-PAGE under nonreducing conditions. The majority of the recombinant autoantibodies were assembled as complete 2H2L heterotetramers. In addition, for rIgA1 and rIgA2, additional bands at higher m.w. were detectable, indicating polymerization and a reduced amount of monomeric rIgA1 or rIgA2. (C) Immunoblotting of recombinant autoantibodies was performed using the same conditions as in (B), as well as detection with peroxidase-conjugated polyclonal anti-human k L chain Ab. The Coomassie staining of rIgA1 and rIgA2 appeared to be weaker overall than that for rIgG1, which may be because of the higher glycosylation content of IgA isotypes. The m.w. standard is indicated on the left. (D) IgA1 and IgA2 recombinant autoantibodies reacted with a 290-kDa band after immunoblotting of dermal extracts, whereas the isotype controls for IgA1 (Iso IgA1) and IgA2 (Iso IgA2) were negative. (E) Human neonatal foreskin cryosections were incubated with the different recombinant autoantibodies and visualized by isotype- and subclass-specific FITC-conjugated Abs. Both rIgA1 and rIgA2 presented a defined reactivity with the DEJ (arrows). As controls, a V-gene matched rIgG1 (31) and serum from a healthy volunteer (NHS) in conjunction with FITC-conjugated polyclonal rabbit anti-human IgG was used. Original magnification 3100.

Article Snippet: For the detection of Abs in cryosections, FITC-conjugated polyclonal goat anti-human IgA (Bio-Rad Laboratories GmbH, Munich, Germany) and polyclonal goat anti-human IgG (Sigma-Aldrich Chemie GmbH, Munich, Germany) were used as secondary Abs.

Techniques: Recombinant, Labeling, SDS Page, Staining, Western Blot, Glycoproteomics, Incubation

Journal: Journal of Biological Chemistry

Article Title: High Resolution Mapping of the Binding Site on Human IgG1 for FcγRI, FcγRII, FcγRIII, and FcRn and Design of IgG1 Variants with Improved Binding to the FcγR

doi: 10.1074/jbc.m009483200

Figure Lengend Snippet: FIG. 1. Binding of anti-IgE E27 IgG1 to human FcgR. A, binding of E27 monomers (solid circles), dimeric (solid squares), and hexameric (open squares) complexes to FcgRIIA-Arg131. B, binding of E27 mono- mers (solid circles), dimeric (solid squares), and hexameric (open squares) complexes to FcgRIIIA-Phe158. Dimers were formed by mixing E27 IgG1 and a F(ab9)2 fragment of goat anti-human k light chain at 1:0.5 molar ratio at 25 °C for 1 h (50). Hexameric complexes (i.e. trim- eric in E27 IgG1) were formed by mixing E27 IgG1 with human IgE in a 1:1 molar ratio at 25 °C for 1 h (36).

Article Snippet: Plates were blocked with assay buffer at 25 °C for 1 h. Serial 3-fold dilutions of monomeric E27 and variants (10.0–0.0045 mg/ml) were added to plates and incubated for 2 h. After washing plates with assay buffer, IgG bound to FcgRI was detected with peroxidase-conjugated F(ab9)2 fragment of goat anti-human F(ab9)2-specific IgG (Jackson ImmunoResearch) or with peroxidase-conjugated protein G (Bio-Rad).

Techniques: Binding Assay