Journal: Nature structural & molecular biology

Article Title: Molecular basis of caspase-1 polymerization and its inhibition by a novel capping mechanism

doi: 10.1038/nsmb.3199

Figure Lengend Snippet: INCA preferentially interacts with caspase-1 CARD . ( a ) Co-expression of His-GFP-ASC CARD and biotinylated INCA showed no significant interaction between INCA and ASC CARD , as indicated by the Western blot. ( b ) Over-stoichiometric amounts of INCA did not inhibit His-GFP-ASC CARD nucleated ASC CARD filament formation. Averages of triplicate experiments were plotted. ( c ) Co-expression of His-GFP-caspase-1 CARD and biotinylated INCA showed interaction between INCA and caspase-1 CARD , as confirmed by the PAGE gel. ( d ) Sub-stoichiometric amounts of INCA inhibited His-GFP-caspase-1 CARD nucleated caspase-1 CARD filament formation. Averages of duplicate experiments were plotted. ( e–f ) Negative-stain electron micrographs of the complex (as shown in panel c at 7 ml) between His-GFP-caspase-1 CARD and biotinylated INCA after streptavidin-gold labeling ( e ) or Ni-NTA-gold labeling ( f ).

Article Snippet: The presence of biotin was confirmed by Western blot using streptavidin-conjugated alkaline phosphatase (streptavidine-AP, Molecular Probes Cat# S921).

Techniques: Expressing, Western Blot, Polyacrylamide Gel Electrophoresis, Staining, Labeling

Journal: Nature structural & molecular biology

Article Title: Molecular basis of caspase-1 polymerization and its inhibition by a novel capping mechanism

doi: 10.1038/nsmb.3199

Figure Lengend Snippet: ICEBERG interacts with caspase-1 CARD by co-mixing. ( a ) Co-expression of His-GFP-caspase-1 CARD and biotinylated ICEBERG. Streptavidin-AP and anti-6×His-tag Western blots were shown to confirm the presence of ICEBERG in complex with His-GFP-caspase-1 CARD after Ni-NTA affinity chromatography. L: Whole cell lysate. S: Soluble fraction. U: Ni-NTA unbound. E: Ni-NTA elute. ( b ) The complex eluted from the void of a Superdex 200 column. ( c–d ) Negative-stain electron micrographs of the complex (as shown in panel b at 7 ml) between His-GFP-caspase-1 CARD and biotinylated ICEBERG after Ni-NTA-gold labeling ( c ) or streptavidin-gold labeling ( d ).

Article Snippet: The presence of biotin was confirmed by Western blot using streptavidin-conjugated alkaline phosphatase (streptavidine-AP, Molecular Probes Cat# S921).

Techniques: Expressing, Western Blot, Affinity Chromatography, Staining, Labeling

Journal: Molecular Biology of the Cell

Article Title: Dissociation of the Tubulin Dimer Is Extremely Slow, Thermodynamically Very Unfavorable, and Reversible in the Absence of an Energy Source

doi: 10.1091/mbc.E01-10-0089

Figure Lengend Snippet: Surface plasmon resonance analysis of biotin-tubulin binding to strepavidin and subsequent dimer dissociation as a result of dilution. (A) The plasmon resonance signal was increased by 954 RU during a 10-min flow of biotin-tubulin in Pi buffer with 12 mM Mg. The almost instantaneous 3000 RU signal change at the start and finish of the flow of the tubulin resulted from a difference in refractive index of the tubulin solution and the buffer. (B) Flow of tubulin- and nucleotide-free buffer resulted in a 445 RU signal decrease; the curve corresponds to a rate constant 14.72 × 10 −5 s −1 . A rate constant equal to 12.35 × 10 −5 s −1 was determined from a Guggenheim plot of the data.

Article Snippet: After three 10-minute washes in PBS, the membrane was incubated for 0.5–16 h with alkaline phosphatase–conjugated streptavidin (Cat no. 21324; Pierce Chemical , Rockford, IL) diluted 46,000-fold in PBS.

Techniques: SPR Assay, Binding Assay, Flow Cytometry

Journal: PLoS ONE

Article Title: High-Threshold Mechanosensitive Ion Channels Blocked by a Novel Conopeptide Mediate Pressure-Evoked Pain

doi: 10.1371/journal.pone.0000515

Figure Lengend Snippet: Biotinylated NMB-1 binds to peripherin-positive DRG neurons. Primary cultures of DRG neurons were stained with biotinylated NMB-1 and streptavidin linked to Cy3. (A,C) or alkaline phosphatase (D). Counter-staining with a monoclonal antibody to peripherin (B) shows in merged figure (C) that nearly all NMB-1 binding neurons express peripherin. Large diameter sensory neurons (E) were negative for NMB-1 binding. No signal was visible when NMB-1 was omitted from the staining procedure.

Article Snippet: Biotinylated NMB-1 was visualized by incubating cultures in alkaline phosphatase-conjugated streptavidin (Chemicon); alkaline phosphatase colour reaction was carried out using BCIP/NBT kit from Vector Laboratories according to the manufacturer's instructions.

Techniques: Staining, Binding Assay

Journal: Clinical and Experimental Immunology

Article Title: The endothelium is an extrahepatic site of synthesis of the seventh component of the complement system

doi: 10.1046/j.1365-2249.2000.01238.x

Figure Lengend Snippet: Immunoblot analysis of C7 secreted by human umbilical vein endothelial cells (HUVEC). C7 (20 ng) purified from cell supernatant by affinity chromatography (lane 1), 0·2 μ l of normal human serum (lane 2) and 0·2 μ l of C7-deficient human serum (lane 3) were subjected to SDS–PAGE on a 10% gel and blotted to nitrocellulose membrane. The immunoblot was then developed with biotin-labelled anti-C7 antibodies followed by streptavidin-alkaline phosphatase. The mol. wt of the C7 band is indicated with reference to the relative mobility of known molecular weight markers.

Article Snippet: Briefly, solid-phase bound MoAb aE11 was used at a dilution of 1/5000 to bind the complex for 1 h at 37°C and, after washing, the bound TCC was evaluated by its reaction with 1/1000 biotin-labelled goat IgG anti-C5 for 1 h at 37°C followed by 30 min incubation at 37°C with 1/4000 alkaline phosphatase conjugated to streptavidin (Sigma-Aldrich).

Techniques: Purification, Affinity Chromatography, SDS Page, Molecular Weight

Journal: Journal of Virology

Article Title: Host Protein Interactions with the 3? End of Bovine Coronavirus RNA and the Requirement of the Poly(A) Tail for Coronavirus Defective Genome Replication

doi:

Figure Lengend Snippet: In vitro binding of PABP to coronavirus 3′ UTR RNAs. In vitro-translated luciferase or PABP was incubated with 1 μg of biotinylated BCV3′UTR RNAs containing poly(A) tails of 1, 5, 10, or 68 A residues (A) or MHV3′UTR RNAs containing poly(A) tails of 0, 5, 10, or > 50 A residues (B). Immobilized streptavidin was added to recover biotinylated RNA complexes, and samples were washed to remove any unbound RNA or protein. Samples were analyzed by SDS-PAGE (8% polyacrylamide). M (lanes 1 and 12) denotes marker and corresponds to the input amount of radiolabeled luciferase (lane 1) or PABP (lane 12) in each reaction. Lanes 2 and 7 represent the level of background protein binding in the absence of biotinylated RNA for luciferase (lane 2) and PABP (lane 7). (A) Lanes 3 to 6, luciferase recovered from interaction with BCV3′UTR RNAs; lanes 8 to 11, PABP recovered from interaction with BCV3′UTR RNAs. (B) Lanes 3 to 6, luciferase recovered from interaction with MHV3′UTR RNAs; lanes 8 to 11, PABP recovered from interaction with MHV3′UTR RNAs.

Article Snippet: One half was processed as described for protein detection, and the other half was incubated with streptavidin conjugated to alkaline phosphatase (Zymed) for 15 min at room temperature in 200 μl of 1× binding buffer.

Techniques: In Vitro, Binding Assay, Luciferase, Incubation, SDS Page, Marker, Protein Binding

Journal: PLoS ONE

Article Title: Natural Pig Plasma Immunoglobulins Have Anti-Bacterial Effects: Potential for Use as Feed Supplement for Treatment of Intestinal Infections in Pigs

doi: 10.1371/journal.pone.0147373

Figure Lengend Snippet: Characterisation of ppIgG. (A) Pig plasma IgG product (ppIgG, 1 mg/ml) was run under reducing conditions on 12% SDS PAGE as described in Materials and Methods. In parallel, Western blotting (WB) was performed on the same sample. The blot was developed with biotinylated rabbit anti-pig IgG F(ab) 2 antibody, followed by alkaline phosphatase-coupled streptavidin (see Materials and Methods ). (B) Wells were coated with formalin-fixed bacteria (Y. ruckeri, S. diarizonae, E. coli O138, O149:F4 and F18+) in separate ELISA plates, and incubated with ppIgG added in serial 2-fold dilutions, and the bound IgG was estimated by using a HRP-conjugated rabbit anti-pig IgG antibody. Data from one typical experiment are presented. (C) ppIgG was tested for reactivity against Escherichia coli and Salmonella diarizonae by competitive ELISA as described ( Materials and Methods ). ppIgG heated at 60°C or 70°C for 1 hour and PBS were used as control samples. The results are presented as ‘% inhibition’ of the signal obtained in the absence of ppIgG (median values (± median ranges)). Two-way ANOVA (subjected to Bonferonni post-test) was used to observe statistical significance between the treated and untreated ppIgG (***: p

Article Snippet: Then, membranes were incubated with polyclonal biotinylated rabbit anti-Pig F(ab)2 IgG (LSBio, Copenhagen, Denmark) diluted 1:10.000 in TBS-T for 1 hour followed by 3x10 min washes in TBS-T before the last incubation with alkaline phosphatase-conjugated streptavidin (DAKO, Glostrup, Denmark) 1:3000 in TBS-T (1 hour).

Techniques: SDS Page, Western Blot, Enzyme-linked Immunosorbent Assay, Incubation, Competitive ELISA, Inhibition