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: Proceedings of SPIE--the International Society for Optical Engineering

Article Title: A method for tethering single viral particles for virus-cell interaction studies with optical tweezers

doi: 10.1117/12.2500050

Figure Lengend Snippet: Biotinylated DNA tether binds streptavidin. (A) Schematic of the double-stranded DNA tether prepared from half lambda DNA that contains functional groups as indicated on both ends of the molecule. (B) A representative force-extension curve for a single DNA tether formed between an anti-digoxigenin coated polystyrene particle and a biotin-coated polystyrene particle. The length of the overstretch transition in this figure measures 5.4 |im, consistent with that of a half-lambda DNA.

Article Snippet: Streptavidin-alkaline phosphatase conjugate (Invitrogen, Catalog #43–4322) at a concentration of 1.5 ug/mL was used to detect biotinylated protein.

Techniques: Lambda DNA Preparation, Functional Assay

Journal: Proceedings of SPIE--the International Society for Optical Engineering

Article Title: A method for tethering single viral particles for virus-cell interaction studies with optical tweezers

doi: 10.1117/12.2500050

Figure Lengend Snippet: Metabolic biotinylation of individual HIV-1 virions. (A) Western blotting of virion preparations with varied inputs of TfR (indicated in the figure) and BirA expression plasmid detected with streptavidin-alkaline phosphatase conjugate. Loaded samples were diluted to have equal p24 concentrations. Lysate from 293T cells transfected with pTfR-AP-IRES-BirA-ER (pTfR) serves as a positive control. (B) Optical trapping virometry results for individual HIV-1 virions: Left: histogram of Alexa 594 fluorescence, proportional to the number of biotinylated transferrin receptors, in individually trapped virions. Right: histogram of EGFP fluorescence in individually trapped virions. N = 28 virions trapped on the same day.

Article Snippet: Streptavidin-alkaline phosphatase conjugate (Invitrogen, Catalog #43–4322) at a concentration of 1.5 ug/mL was used to detect biotinylated protein.

Techniques: Western Blot, Expressing, Plasmid Preparation, Transfection, Positive Control, Fluorescence

Journal: Plant Physiology

Article Title: A Multisubunit Acetyl Coenzyme A Carboxylase from Soybean 1

doi:

Figure Lengend Snippet: Analysis of column fractions from total soybean seed extracts after chromatography through antibody-affinity columns. Lanes 1, Protein-gel blot probed with streptavidin-alkaline phosphatase conjugate; lanes 2, anti-BCCP probe; lanes 3, anti-BC probe; lanes 4, anti-α-CT probe; lanes 5, anti-β-CT probe. Gly eluates from each column (indicated above the lanes) were separated on 9% SDS-PAGE and transferred to PVDF membranes. Each membrane was subsequently cut into strips and probed with each ACCase antibody.

Article Snippet: Biotinylated proteins were detected with a streptavidin-alkaline phosphatase conjugate (Boehringer Mannheim) diluted 1:2,000.

Techniques: Chromatography, Western Blot, SDS Page

Journal: Plant Physiology

Article Title: A Multisubunit Acetyl Coenzyme A Carboxylase from Soybean 1

doi:

Figure Lengend Snippet: ) at 0.05 mL/min. Fractions (0.75 mL) were collected, and a high-molecular-mass gel-filtration calibration kit (Pharmacia) was used to calibrate the column. Eluted protein fractions were separated on SDS-PAGE, blotted to nylon membranes, and analyzed with different antibodies as indicated. strept, Streptavidin-alkaline phosphatase conjugate. The results from the protein-gel blot are aligned with the chromatographic profile. Approximate positions of electrophoretic molecular-mass markers are shown at the right of the protein-blot panels. The position of Blue Dextran 2000 (BD) and other markers used to calibrate the column are shown above the elution profile. The markers were thryoglobulin (667 kD), ferritin (440 kD), catalase (232 kD), and aldolase (158 kD).

Article Snippet: Biotinylated proteins were detected with a streptavidin-alkaline phosphatase conjugate (Boehringer Mannheim) diluted 1:2,000.

Techniques: Filtration, SDS Page, Western Blot