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.

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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.