rabbit anti secretogranin ii  (Thermo Fisher)

Journal:

Article Title: Role of Secretory Carrier Membrane Protein SCAMP2 in Granule Exocytosis

doi: 10.1091/mbc.E02-03-0136

Figure Lengend Snippet: E peptide inhibits stimulated release of 35S-labeled secretogranin from permeabilized PC12 cells. Cells in 24-well plates were labeled overnight with [35S]sulfate, chased, and permeabilized with SLO. They were then equilibrated 30 min on ice with or without (control) EGTA-buffered Ca2+ (10 μM) and with or without E peptide. Secretion of 35S-labeled SG was evaluated as in MATERIALS AND METHODS. (A) E peptides from normal SCAMP2, normal SCAMP1, and structural variants of SCAMP2 in which residues 1, and 2 and 3 together, were changed from C to A and WY to AA, respectively, were compared at a concentration of 100 μM. Stimulated secretion of 35S-labeled secretogranin ranged from 35 to 45% of total above an unstimulated secretion of 1–2% of total. The inset presents a sample autoradiograph that demonstrates clearly the effects of the peptide on secretion. (B) Dose-response curve for inhibition of secretion by E peptide of SCAMP2. The results shown are normalized to stimulated secretion from peptide-free samples from three independent experiments and are plotted as mean ± SEM.

Article Snippet: Rabbit anti-secretogranin II was from Biodesign International (Saco, ME); portions of this antibody were biotinylated using NHS-biotin (Pierce Endogen, Rockford, IL) according to the manufacturer's instructions.

Techniques: Labeling, Concentration Assay, Autoradiography, Inhibition

Journal:

Article Title: Role of Secretory Carrier Membrane Protein SCAMP2 in Granule Exocytosis

doi: 10.1091/mbc.E02-03-0136

Figure Lengend Snippet: .Localization of SCAMPs 1 and 2 in PC12 cells. (A and B) Double-label immunofluorescence images showing the distributions of the two SCAMPs (stained with antibodies 1ω and 2τ and Alexa 488-tagged secondary antibody) compared with secretogranin (SG; stained with biotinylated anti-SG and avidin-Alexa 594). A digitally deconvolved section (0.2 μm) including an unstained nuclear profile is shown in each case. Bar, 10 μm. (C) Distribution of SCAMPs compared with secretogranin in large dense-core vesicles purified by gradient centrifugation. Plots of SG and SC2 are intensity profiles of bands from Western blots probed with anti-SG and anti-SC2 (2τ), detected with 125I-labeled secondary antibody, and analyzed by phosphorimaging. The accompanying Western blots show the distribution of SG and of SCAMPs 1–3 as detected by mAb 7C12 and enhanced chemiluminescence. (D) Distributions of SCAMP2 and SG as observed on plasma membrane sheets (Lang et al., 2001 ) using double-label immunofluorescence (antibodies as in B). The bar corresponds to 5 μm. (E) Immunogold-stained electron microscopic images showing multiply labeled SCAMP2 foci (open arrowheads) on plasma membranes torn from the upper cellular surface. Primary antibody 2τ was used. The fourth panel, labeled C, is a negative control showing an occasional solitary gold particle when primary antibody staining was omitted. * identifies a clathrin-coated pit; bar, 0.2 μm.

Article Snippet: Rabbit anti-secretogranin II was from Biodesign International (Saco, ME); portions of this antibody were biotinylated using NHS-biotin (Pierce Endogen, Rockford, IL) according to the manufacturer's instructions.

Techniques: Immunofluorescence, Staining, Avidin-Biotin Assay, Purification, Gradient Centrifugation, Western Blot, Labeling, Negative Control

Journal:

Article Title: Role of Secretory Carrier Membrane Protein SCAMP2 in Granule Exocytosis

doi: 10.1091/mbc.E02-03-0136

Figure Lengend Snippet: (A–F) Comparison of the distributions of SCAMP2, syntaxin1, and secretogranin by triple-immunostaining of plasma membrane sheets prepared according to Lang et al. (2001) . (A–C) Individual images of a sheet stained with anti-SCAMP2 (2τ)/Alexa 488–tagged secondary antibody (A), biotinylated anti-SG/avidin-pacific blue (B), and anti-syntaxin 1 (mAb HPC1)/Alexa 594–tagged secondary antibody (C). (D) Merged image of A–C. Open arrowheads identify several examples of SCAMP2 overlapping SG but not syntaxin 1, and filled arrowheads identify overlapping SCAMP2, SG, and syntaxin 1. (E and F) Quantitative comparison of the distributions of SCAMP2 (E) and SG (F) with respect to the other labeled antigens obtained from counts of five separate plasma membrane sheets differing from the one shown in the figure. D: Bar, 5 μm.

Article Snippet: Rabbit anti-secretogranin II was from Biodesign International (Saco, ME); portions of this antibody were biotinylated using NHS-biotin (Pierce Endogen, Rockford, IL) according to the manufacturer's instructions.

Techniques: Triple Immunostaining, Staining, Avidin-Biotin Assay, Labeling

Journal:

Article Title: Role of Secretory Carrier Membrane Protein SCAMP2 in Granule Exocytosis

doi: 10.1091/mbc.E02-03-0136

Figure Lengend Snippet: Expression and localization of myc-tagged SCAMP2 in Tet-regulated PC12 cells. Dox was added at 48 h after transfection, and cells were double-labeled with mouse anti-myc antibody and anti-secretogranin. Deconvolved images were prepared from five cells of each type expressing wild-type SCAMP2, mutant A (C→A), or mutant B (W→A). Representative optical sections (0.2 μm) including profiles of nuclei are shown. Bar, 10 μm.

Article Snippet: Rabbit anti-secretogranin II was from Biodesign International (Saco, ME); portions of this antibody were biotinylated using NHS-biotin (Pierce Endogen, Rockford, IL) according to the manufacturer's instructions.

Techniques: Expressing, Transfection, Labeling, Mutagenesis