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.

Anti-Otx1 + Otx2 antibody (ab21990) at 1 µg/ml + OTX1 (Human) - Recombinant Protein at 0.1 µg

Secondary
Goat Anti-Rabbit IgG H&L (HRP) preadsorbed (ab97080) at 1/5000 dilution

Developed using the ECL technique.

Performed under reducing conditions.

Predicted band size: 31.6 kDa
Observed band size: 74 kDa why is the actual band size different from the predicted?



This Otx1 recombinant protein has a GST tag causing it to run at ~74kDa.

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.

All lanes : Anti-Otx1 + Otx2 antibody (ab21990) at 1 µg/ml

Lane 1 : Y79 Lysate
Lane 2 : Y79 Lysate with Otx2 peptide (ab24347) at 1 µg/ml

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Alexa Fluor Goat polyclonal to Rabbit IgG at 1/10000 dilution

Predicted band size: 31.6 kDa
Observed band size: 37 kDa why is the actual band size different from the predicted?
Additional bands at: 28 kDa (possible cleavage fragment), 28 kDa (possible isoform), 28 kDa (possible non-specific binding)

The image shows staining of Otx2 in human embryonic stem cells differentiated into neuroectoderm. As would be expected, staining is nuclear.