The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
Use at an assay dependent concentration. PubMed: 20872847
Use a concentration of 1 µg/ml. Detects a band of approximately 9 kDa (predicted molecular weight: 9 kDa).
Abcam recommends using 1-3% Milk as the blocking agent.
1/1000. Perform heat mediated antigen retrieval via the microwave method before commencing with IHC staining protocol.
Use at an assay dependent concentration. PubMed: 21742036
May participate in the maintenance of segment identity in the hindbrain and pituitary development, and maturation or maintenance of the overall structure of the nervous system. May function as a regulatory subunit of ion channels.
Belongs to the neuronatin family.
Abundant in 18-24 week old fetal brain. Postnatally its expression decline and only minimal levels were present in adulthood.
Anti-Neuronatin antibody (ab27266) at 1 µg/ml + Brain (Mouse) Tissue Lysate - normal tissue, 0 days old (blocked with 3% Milk) at 50 µg
Secondary Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/10000 dilution Developed using the ECL technique
Performed under reducing conditions.
Predicted band size : 9 kDa Observed band size : 9 kDa
Exposure time : 12 minutes
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Neuronatin antibody (ab27266)This image is courtesy of an anonymous Abreview
ab27266 at 1/1000 staining human pituitary cells by IHC-P
The antibody was tested on normal human pituitary slide sections (formalin fixed/paraffin embedded). This gave good staining over a range of concentrations with a microwave/pressure antigen retrieval step. Staining was strongly associated with the cell membrane and blocked by incubating with the neuronatin antibody with the peptide which the antibody was raised against.
ICC/IF image of ab27266 stained SKNSH cells. The cells were 100% methanol fixed (5 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab27266, 5µg/ml) overnight at +4°C. The secondary antibody (green) was ab96899, DyLight® 488 goat anti-rabbit IgG (H+L) used at a 1/250 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.
Immunohistochemistry (PFA perfusion fixed frozen sections) - Anti-Neuronatin antibody (ab27266)Image from Wagnon JL et al., PLoS Genet 8(11): e1003067.. Fig 6.; doi: 10.1371/journal.pgen.1003067. Epub 2012 Nov 29.
Immunohistochemical analysis of PFA-perfusion fixed mouse brain tissue, staining Neuronatin with ab27266.
Tissue was post-fixed in 4% paraformaldehyde overnight at 4°C. Sections were incubated in a blocking buffer of 0.3% Triton-X-100, 1% BSA and 10% NGS in PBS for 2 hours at room temperature, before incubating with primary antibody (1/100) for 2 days at 4°C. An AlexaFluor®-conjugated anti-rabbit IgG (1/1000) was used as the secondary antibody.