Anti-Otx1 + Otx2 antibody [EPR3347] - ChIP Grade (ab92515) at 1/2500 dilution (purified) + Y79 cell lysate at 10 µg

Secondary
HRP goat anti-rabbit IgG (H+L) at 1/20000 dilution

Predicted band size: 32 kDa
Observed band size: 32 kDa

Blocking buffer: 5% NFDM/TBST
Dilution buffer: 5% NFDM/TBST

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human retina tissue labelling Otx1 + Otx2 with purified ab92515 at a dilution 1/500. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9 (ab93684). ab97051, a HRP-conjugated goat anti-rabbit IgG (H+L) was used as the secondary antibody (1/500). Counterstained with hematoxylin.

Nuclear staining on inner nuclear layer and outer nuclear layer of the human retina.

Chromatin was prepared from mouse stem cell D3 according to the Abcam X-ChIP protocol. Cells were fixed with formaldehyde for 10 minutes. The ChIP was performed with 25µg of chromatin, 5µg of ab92515 (blue), and 20µl of Anti rabbit IgG sepharose beads. 5μg of rabbit normal IgG was added to the beads control (yellow). The immunoprecipitated DNA was quantified by real time PCR (Sybr green approach).

Otx1 + Otx2 was immunoprecipitated from Y79（Human retinoblastoma retinoblastoma）whole cell lysate with ab92515 at 1/20 dilution. Western blot was performed from the immunoprecipitate using ab92515 at 1/500 dilution. Secondary antibody details (ab131366), was used as secondary antibody at 1/1000 dilution.

Blocking/Diluting Buffer and concentration: 5% NFDM /TBST.

Lanes 1-2 : Anti-Otx1 + Otx2 antibody [EPR3347] - ChIP Grade (ab92515) at 1/500 dilution (purified)
Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab92515

Lane 1 : Y79 (Human retinoblastoma retinoblastoma) whole cell lysate
Lanes 2-3 : Y79 whole cell lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : VeriBlot for IP Detection Reagent (HRP) (ab131366) at 1/1000 dilution

Anti-Otx1 + Otx2 antibody [EPR3347] - ChIP Grade (ab92515) at 1 µg/ml (unpurified) + OTX1 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: 32 kDa
Observed band size: 75 kDa why is the actual band size different from the predicted?

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human retinoblastoma tissue labelling Otx1 + Otx2 with purified ab92515 at a dilution 1/500. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9 (ab93684). ab97051, a HRP-conjugated goat anti-rabbit IgG (H+L) was used as the secondary antibody (1/500). Counterstained with hematoxylin.

Nuclear staining on tumor cells of human retinoblastoma.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of mouse retina tissue labelling Otx1 + Otx2 with purified ab92515 at a dilution 1/500. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9 (ab93684). ab97051, a HRP-conjugated goat anti-rabbit IgG (H+L) was used as the secondary antibody (1/500). Counterstained with hematoxylin.

Nuclear staining on nuclear layer of the mouse retina.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of mouse E14.5 choroid plexus tissue labelling Otx1 + Otx2 with purified ab92515 at a dilution 1/500. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9 (ab93684). ab97051, a HRP-conjugated goat anti-rabbit IgG (H+L) was used as the secondary antibody (1/500). Counterstained with hematoxylin.

Nuclear staining on mouse E14.5 choroid plexus.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of rat E14.5 choroid plexus tissue labelling Otx1 + Otx2 with purified ab92515 at a dilution 1/1000. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9 (ab93684). ab97051, a HRP-conjugated goat anti-rabbit IgG (H+L) was used as the secondary antibody (1/500). Counterstained with hematoxylin.

Nuclear staining on rat E14.5 choroid plexus.

Immunohistochemical staining of paraffin embedded human cerebral cortex with purified ab92515 at a working dilution of 1/250. The secondary antibody used is HRP goat anti-rabbit IgG H&L (ab97051) at 1/500. The sample is counter-stained with hematoxylin. Antigen retrieval was perfomed using Tris-EDTA buffer, pH 9.0. PBS was used instead of the primary antibody as the negative control, and is shown in the inset.