Anti-Neuronatin antibody (ab27266)

Anti-Neuronatin antibody (ab27266) at 1 µg/ml + Mouse brain tissue lysate - total protein (0 days) (ab7188) at 20 µg

Secondary
IRDye 680 Conjugated Goat Anti-Rabbit IgG (H+L) at 1/15000 dilution

Performed under reducing conditions.

Predicted band size: 9 kDa

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

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.

See Abreview

Anti-Neuronatin antibody (ab27266) at 1 µg/ml + Mouse brain tissue lysate - total protein (0 days) (ab7188) at 10 µg

Secondary
Goat polyclonal to Rabbit IgG - H&L - Pre-Adsorbed (HRP) at 1/3000 dilution

Performed under reducing conditions.

Predicted band size: 9 kDa
Observed band size: 10 kDa (why is the actual band size different from the predicted?)
Additional bands at: 70-75 kDa. We are unsure as to the identity of these extra bands.


Exposure time: 5 minutes

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.

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.