Anti-nNOS (neuronal) (phospho S847) antibody

• IHC-FoFr

Collaborator

Immunohistochemistry (PFA perfusion fixed frozen sections) - Anti-nNOS (neuronal) (phospho S847) antibody (AB16650)

Immunostaining using Rabbit polyclonal to nNOS (neuronal) (phospho S847) (ab16650) on rat brain tissue sections (30 micron free floating). ab16650 was used at a dilution of 1/3000 and incubated for 18 hours at RT (in PBS triton 0.3%). Secondary Antibody Goat anti-rabbit alexa Fluor 488 was used at a dilution of 1/1000. The image shows cytoplasmic staining of CNS neurons with ab16650 in naïve rats; the staining being observed in the soma and processes of these neurons. The staining was quenched by pre-incubation with peptide against phospho S847 (ab16981), but not by the control peptide (ab57047) indicating that ab16650 is specific for nNos phosphorylated at S847. Protocol : Rats were perfused-fixed with 4% paraformaldehyde. Tissues were post-fixed overnight in the same fixative and then cryoprotected in 20% sucrose overnight. Following embedding in OCT and freezing, tissues were cut and immunostained using the 'free floating’ technique.

This image is courtesy of Sophie Pezet, CNRS, Paris, France

• WB

Project

Western blot - Anti-nNOS (neuronal) (phospho S847) antibody (AB16650)

All lanes:

Western blot - Anti-nNOS (neuronal) (phospho S847) antibody (ab16650) at 1 µg/mL

Lane 1:

Forebrain (Mouse) Tissue Lysate at 20 µg

Lane 2:

Spinal Cord (Mouse) Tissue Lysate at 20 µg

Lane 3:

HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate (negative control) at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-preadsorbed-ab97080'>ab97080</a>) at 1/5000 dilution

Predicted band size: 160 kDa

Observed band size: 190 kDa

true

Exposure time: 2min

• WB

Ap

Western blot - Anti-nNOS (neuronal) (phospho S847) antibody (AB16650)

This blot was produced using a 3-8% Tris Acetate gel under the TA buffer system. The gel was run at 150V for 60 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour using 2% Bovine Serum Albumin before being incubated with ab16650 overnight at 4°C. Antibody binding was detected using an anti-rabbit antibody conjugated to HRP, and visualised using ECL development solution ab133406.

All lanes:

Western blot - Anti-nNOS (neuronal) (phospho S847) antibody (ab16650) at 1 µg/mL

Lane 1:

Forebrain (Mouse) Tissue Lysate at 20 µg

Lane 2:

Spinal Cord (Mouse) Tissue Lysate at 20 µg

Lane 3:

HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate (negative control) at 20 µg

Lane 4:

Forebrain (Mouse) Tissue Lysate at 20 µg with Mouse nNOS (neuronal) (phospho S847) peptide

Lane 5:

Spinal Cord (Mouse) Tissue Lysate at 20 µg with Mouse nNOS (neuronal) (phospho S847) peptide

Lane 6:

HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate (negative control) at 20 µg with Mouse nNOS (neuronal) (phospho S847) peptide

Lane 7:

Forebrain (Mouse) Tissue Lysate at 20 µg with Mouse nNOS (neuronal) (unmodified) peptide

Lane 8:

Spinal Cord (Mouse) Tissue Lysate at 20 µg with Mouse nNOS (neuronal) (unmodified) peptide

Lane 9:

HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate (negative control) at 20 µg with Mouse nNOS (neuronal) (unmodified) peptide

Secondary

All lanes:

Goat polyclonal to Rabbit IgG - H&L - Pre-Adsorbed (HRP) at 1/50000 dilution

Predicted band size: 160 kDa

Observed band size: 190 kDa,55 kDa

true

Exposure time: 4min

• WB

Collaborator

Western blot - Anti-nNOS (neuronal) (phospho S847) antibody (AB16650)

Lanes 1 - 3:

Western blot - Anti-nNOS (neuronal) (phospho S847) antibody (ab16650) at 1 µg/mL

Lane 4:

nNOS antibody at 1/2500 dilution

Lanes 1 and 4:

mouse forebrain at 20 µg

Lane 2:

mouse forebrain at 20 µg with Mouse nNOS (neuronal) (phospho S847) peptide (<a href='/en-us/products/unavailable/mouse-nnos-neuronal-phospho-s847-peptide-ab16981'>ab16981</a>)

Lane 3:

mouse forebrain at 20 µg with corresponding unmodified nNOS (neuronal) peptide

Predicted band size: 160 kDa

Observed band size: 160 kDa

false

This image is courtesy of Chris Anderson, Wellcome Trust Sanger Institute, United Kingdom