Journal: Scientific Reports

Article Title: A tool kit for rapid cloning and expression of recombinant antibodies

doi: 10.1038/srep05885

Figure Lengend Snippet: (A–B) Flow cytometric histograms showing fluorescent intensity of cells incubated with purified IgG/IgE antibodies plus secondary FITC-labelled goat anti-human IgG/IgE (open) and cells with secondary antibody only (filled) against the number of cells. (A) Flow cytometric assessment of anti-CSPG4 IgG 1 /IgE antibodies shows specific binding to native CSPG4 antigen present on the cell surface of A375 melanoma cells and no binding above background to primary melanocytes. (B) Fc regions of anti-CSPG4 IgG 1 and 102.1F10 IgG 4 isotypes demonstrate effector-binding to U937 monocytic cell line, expressing human Fcγ receptors. The IgE antibody isotypes also bind similarly to RBL SX38 mast cells, expressing human FcεR1 receptor. (C) Immunofluorescence staining of A375 cells confirms specific binding of anti-CSPG4 IgG 1 /IgE and no background binding with isotype control hapten specific NIP-IgG 1 /IgE detected by goat anti-human IgG/IgE-FITC. (D) Grass pollen allergen specificity is confirmed by anti-human sandwich ELISA, showing specific binding of recombinant 102.1F10 IgG 4 /IgE and original patient serum to the ELISA plate-bound Phl p 7 allergen and no binding above background with unspecific human myeloma IgG 4 and MOv18 IgE isotype controls.

Article Snippet: Primary human epidermal melanocytes (PCS-200-012, ATCC) were grownin Dermal Cell Basal Medium (PCS-200-030, ATCC) and supplemented with the Melanocyte Growth Kit (PCS-200-041, ATCC).

Techniques: Incubation, Purification, Binding Assay, Expressing, Immunofluorescence, Staining, Control, Sandwich ELISA, Recombinant, Enzyme-linked Immunosorbent Assay

Journal: Toxicology and applied pharmacology

Article Title: Monoubiquitinated γ-H2AX: abundant product and specific biomarker for non-apoptotic DNA double-strand breaks

doi: 10.1016/j.taap.2018.07.007

Figure Lengend Snippet: Panels A-C used antibodies #1. (A) Impact of gel polyacrylamide percentage and transfer conditions to PVDF membranes. Treatments of H460 cells: 6 h with 3 mM hydroxyurea (HU), 6 or 16 h with 5 μM aphidicolin (Aphi) or 1 h with 5 μM bleomycin (Bleo). All γ-H2AX images had 5 s film exposures. (B) Ethanol effect in semi-dry transfer. IMR90 cells were treated with 40 μM etoposide (Eto) for 1 h. (C) Comparison of wet (pH 8.2 and pH 8.8) and semi-dry transfers using 12% ethanol-containing buffers (10 s film exposures for all γ-H2AX images). IMR90 cells were treated with 40 μM etoposide for 1 h. (D) Effectiveness of different antibodies using semi-dry transfer with 12% ethanol. Normal human keratinocytes were treated for 1 h with 30 μM etoposide or 0.5 μM bleomycin. Antibodies #1–3: 1:1000 dilutions and 5 s exposures, antibody #4: 1:1000 dilution and 60 s exposure. (E) Comparison of antibodies #1 and #5 (both at 1:1000 dilutions) in H460 cells treated with bleomycin for 1 h. (F) γ-H2AX blot (Ab#1) in control and H2AX-depleted IMR90 cells treated with 100 μM etoposide for 1h.

Article Snippet: IMR90, WI38, normal human neonatal keratinocytes (PCS-200–010), H460, A549, U2OS, mouse embryonic fibroblasts (MEFs, SCRC-1040), telomerase-immortalized human bronchial epithelial HBEC3 (CRL-4051), Daudi, HCT116, Ramos, Raji and CA46 were obtained from ATCC.

Techniques: Comparison, Control

Journal: BMC Bioinformatics

Article Title: Structure and sequence analyses of Bacteroides proteins BVU_4064 and BF1687 reveal presence of two novel predominantly-beta domains, predicted to be involved in lipid and cell surface interactions

doi: 10.1186/s12859-014-0434-7

Figure Lengend Snippet: Structures of the N-terminally truncated Bacteroides proteins BVU_4064 and BF1687 (PDB codes 3kog and 3g3l, respectively). The N-terminal domain (in slate blue color) and the C-terminal domain (in orange color) of the 3kog structure show significant similarities with the corresponding domains of 3g3l structure (N and C terminal domains shown in pale cyan and wheat colors respectively). In contrast, the region connecting the domains (in green) is clearly different in the two structures: a short linker in 3kog, an extended 4-helix insertion and one extra strand that is added to the C-terminal domain in 3g3l. A histidine-rich region present at the C-terminus in both of our proteins is found ordered only in the 3kog structure (see box with text in the Figure).

Article Snippet: The crystal structures of the N-terminally truncated Bacteroides proteins BVU_4064 ( Bacteroides vulgatus strain ATCC 8482, JCSG target ID: 393242, GenBank accession: YP_001301288.1, PDB code: 3kog) and BF1687 ( Bacteroides fragilis strain NCTC 9343, JCSG target ID: 393243, Gene Bank accession: YP_211325.1, PDB code: 3g3l) have been determined to 1.85 Å and 2.2 Å resolution, using MAD and SAD phasing methods respectively as described in the section.

Techniques:

Journal: BMC Bioinformatics

Article Title: Structure and sequence analyses of Bacteroides proteins BVU_4064 and BF1687 reveal presence of two novel predominantly-beta domains, predicted to be involved in lipid and cell surface interactions

doi: 10.1186/s12859-014-0434-7

Figure Lengend Snippet: Superposition of 3kog and 3g3l structures. (A) Corresponding domains (colored in slate blue and pale cyan for N-terminal domains; orange and wheat for C-terminal domains; linker region in green) in the two structures superimpose fairly well with an overall RMSD of 3.7 Å for the 166 equivalent positions in the rigid-body alignment . (B) Stereo view of N and C terminal domains shown separately with linker regions removed to highlight the structural similarity.

Article Snippet: The crystal structures of the N-terminally truncated Bacteroides proteins BVU_4064 ( Bacteroides vulgatus strain ATCC 8482, JCSG target ID: 393242, GenBank accession: YP_001301288.1, PDB code: 3kog) and BF1687 ( Bacteroides fragilis strain NCTC 9343, JCSG target ID: 393243, Gene Bank accession: YP_211325.1, PDB code: 3g3l) have been determined to 1.85 Å and 2.2 Å resolution, using MAD and SAD phasing methods respectively as described in the section.

Techniques:

Journal: BMC Bioinformatics

Article Title: Structure and sequence analyses of Bacteroides proteins BVU_4064 and BF1687 reveal presence of two novel predominantly-beta domains, predicted to be involved in lipid and cell surface interactions

doi: 10.1186/s12859-014-0434-7

Figure Lengend Snippet: Structural similarities of the N-terminal domains. (A-F) Pre-albumin-like fold of the N-terminal domains in 3kog and 3g3l structures that is also present as a cell adhesion modules in several proteins belonging to the Transthyretin superfamily. (G) Alignment between the lipoprotein signal sequences present at the N-terminus of BVU_4064 and BF1687. The arrow points to the conserved CYS residue in the consensus sequence for the protein family PF12985.

Article Snippet: The crystal structures of the N-terminally truncated Bacteroides proteins BVU_4064 ( Bacteroides vulgatus strain ATCC 8482, JCSG target ID: 393242, GenBank accession: YP_001301288.1, PDB code: 3kog) and BF1687 ( Bacteroides fragilis strain NCTC 9343, JCSG target ID: 393243, Gene Bank accession: YP_211325.1, PDB code: 3g3l) have been determined to 1.85 Å and 2.2 Å resolution, using MAD and SAD phasing methods respectively as described in the section.

Techniques: Residue, Sequencing

Journal: BMC Bioinformatics

Article Title: Structure and sequence analyses of Bacteroides proteins BVU_4064 and BF1687 reveal presence of two novel predominantly-beta domains, predicted to be involved in lipid and cell surface interactions

doi: 10.1186/s12859-014-0434-7

Figure Lengend Snippet: Structural similarities of the C-terminal domain of 3kog and 3g3l with bacterial pore-forming toxins. The region shown in red is implicated in membrane insertion in the pore-forming toxins [epsilon toxin (PDB code: 1uyj) and aerolysin (PDB code: 1z52)] and in the hemolytic lectin (PDB code 1w3g). In both 3kog and 3g3l this region corresponds to a helical insertion.

Article Snippet: The crystal structures of the N-terminally truncated Bacteroides proteins BVU_4064 ( Bacteroides vulgatus strain ATCC 8482, JCSG target ID: 393242, GenBank accession: YP_001301288.1, PDB code: 3kog) and BF1687 ( Bacteroides fragilis strain NCTC 9343, JCSG target ID: 393243, Gene Bank accession: YP_211325.1, PDB code: 3g3l) have been determined to 1.85 Å and 2.2 Å resolution, using MAD and SAD phasing methods respectively as described in the section.

Techniques: Membrane