Our Abpromise guarantee covers the use of ab21990 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
|IHC-Fr||Use a concentration of 0.8 µg/ml.|
|ICC/IF||Use a concentration of 0.8 - 4 µg/ml.|
|ChIP||Use at an assay dependent concentration. PubMed: 18057103|
|WB||Use a concentration of 1 µg/ml. Detects a band of approximately 37 kDa (predicted molecular weight: 31.6 kDa).|
|IHC-P||Use at an assay dependent concentration. PubMed: 20150232|
|IP||Use a concentration of 5 µg/ml.|
Function: Probably plays a role in the development of the brain and the sense organs. Can bind to the BCD target sequence (BTS): 5'-TCTAATCCC-3'.
Tissue specificity: Expressed in brain.
Disease: Defects in OTX2 are the cause of microphthalmia syndromic type 5 (MCOPS5) [MIM:610125]. Microphthalmia is a clinically heterogeneous disorder of eye formation, ranging from small size of a single eye to complete bilateral absence of ocular tissues. Up to 80% of cases of microphthalia occur in association with syndromes that include non-ocular abnormalities. MCOPS5 patients manifest unilateral or bilateral microphthalmia/clinical anophthalmia and variable additional features including coloboma, microcornea, cataract, retinal dystrophy, hypoplasia or agenesis of the optic nerve, agenesis of the corpus callosum, developmental delay, joint laxity, hypotonia, and seizures.
Similarity: Belongs to the paired homeobox family. Bicoid subfamily. Contains 1 homeobox DNA-binding domain.
Developmental stage: Embryo.
ab21990 staining Otx2 in embryonic day 14.5 (E14.5) mouse eye by Immunohistochemistry (formalin fixed, paraffin embedded sections).
The embryo was fixed in 10% neutral buffered formalin overnight at room temperature, then paraffin-embedded and sectioned at 4µm. Following deparaffinization and antigen retrieval in a rice steamer in 10mM sodium citrate (pH 6.0), the sections were blocked with 5% normal goat serum for half an hour at room temperature. ab21990 was diluted in PBS at 1/100, and incubated overnight at 4°C. The secondary was goat anti-rabbit Alexa®568, diluted in PBS with 1.5% normal goat serum. The mounting medium contained the nuclear stain DAPI (blue).
Mouse E6 embryos were sectioned and then fixed in paraformaldehyde. Antigen retrieval was performed using citric acid and the sections permeablized and blocked for 1 hour in serum. Embryos were stained with ab21990 (1/2500) diluted in 10% serum, 1% ovalbumin in PBSTween for 14 hours at 4°C. They were then washed and stained with a goat anti-rabbit biotin conjugated antibody. After using a biotinylated secondary, ABC and then DAB was used for color development.
Otx1 +2 was immunoprecipitated using 0.5mg Y79 whole cell extract, 5µg of Rabbit polyclonal to Otx1 +2 and 50µl of protein G magnetic beads (+). No antibody was added to the control (-).
The antibody was incubated under agitation with Protein G beads for 10min, Y79 whole cell extract lysate diluted in RIPA buffer was added to each sample and incubated for a further 10min under agitation.
Proteins were eluted by addition of 40µl SDS loading buffer and incubated for 10min at 70°C; 10µl of each sample was separated on a SDS PAGE gel, transferred to a nitrocellulose membrane, blocked with 5% BSA and probed with ab21990.
Secondary: Mouse monoclonal [SB62a] Secondary Antibody to Rabbit IgG light chain (HRP) (ab99697).
Band: 37kDa; Otx1 +2, non specific - as present in control (lane 2); 37kDa: We are confident this was due to slight lane contamination and the band seen in the IP lane is our target of interest.