antibody primary anti-stx-1a 1b11-11a8  (Novus Biologicals)

Journal:

Article Title: Functional and Biochemical Analysis of the C2 Domains of Synaptotagmin IV

doi:

Figure Lengend Snippet: Distinct Syt–Stx interactions exist for Syt I and Syt IV isoforms. (A) The indicated recombinant GST fusion proteins were immobilized on glutathione–agarose and incubated with 800 μg of rat brain synaptosomes in the absence (−) or presence (+) of 3 mM calcium. Protein complexes were isolated, fractionated by SDS-PAGE, and examined by Western analysis. Stx 1a binding was detected with HPC-1, followed by enhanced chemiluminescence. One hundred nanograms of soluble Stx 1a (Con) were used as a Western control. (B) Ten micrograms of immobilized recombinant Syt I and IV C2A domains were incubated with 100 nM soluble recombinant Stx 1a (amino acids 4–266) or HA-tagged Stxs 2 (amino acids 4–264), 3 (amino acids 4–264), or 4 (amino acids 1–268), and the protein complexes were examined by Western analysis. Stx 1a binding was detected with the anti-Stx 1a antibody HPC-1. Binding of HA-tagged Stxs 2–4 was detected with the anti-HA antibody 12CA5. No binding was detected with GST alone (Con).

Article Snippet: After blocking, the filters were incubated in buffer A for 1 h at room temperature with primary antibodies: anti-Stx 1a mAb (HPC-1) diluted 1:10,000, anti-HA antibody (12CA5) diluted 1:3000, affinity-purified anti-Syt IV antibody (1:1000), and anti-GST mAb (1:1000) (Zymed, San Francisco, CA).

Techniques: Recombinant, Incubation, Isolation, SDS Page, Western Blot, Binding Assay

Journal:

Article Title: Functional and Biochemical Analysis of the C2 Domains of Synaptotagmin IV

doi:

Figure Lengend Snippet: The polybasic motif in the C2A domain of Syt IV functions in calcium-regulated secretion. (A) The indicated recombinant GST fusion proteins were immobilized on glutathione–agarose and incubated with 800 μg of rat brain synaptosomes in the presence (+) or absence (−) of 3 mM calcium. Protein complexes were isolated, fractionated by SDS-PAGE, and examined by Western analysis. Stx 1a binding was detected with HPC-1, followed by enhanced chemiluminescence. One hundred nanograms of soluble Stx 1a (Con) were used as a Western control. (B) NGF-differentiated PC12 cells were coinjected with Texas Red–conjugated dextran and the indicated soluble recombinant Syt IV or Nedd4 fragments. Control cells were injected with Texas Red–conjugated dextran only (Texas Red) or coinjected with a control GST extract (GST). One hour after microinjection, the cells were K+ depolarized in the presence of calcium, and DβH surface immunoreactivity was detected with a fluorescein-labeled secondary antibody. The numbers of individual fluorescent particles were counted, regardless of their size, and are presented as a percent of the total number of cells injected. Data are shown as the mean of two independent experiments ± SD with the total number of injected cells (n). Significant differences (p ≤ 0.01, χ2 analysis) between experimental and control treatments are indicated with an asterisk.

Article Snippet: After blocking, the filters were incubated in buffer A for 1 h at room temperature with primary antibodies: anti-Stx 1a mAb (HPC-1) diluted 1:10,000, anti-HA antibody (12CA5) diluted 1:3000, affinity-purified anti-Syt IV antibody (1:1000), and anti-GST mAb (1:1000) (Zymed, San Francisco, CA).

Techniques: Recombinant, Incubation, Isolation, SDS Page, Western Blot, Binding Assay, Injection, Labeling