Anti-Otx1 + Otx2 antibody

10 product images

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  Immunoprecipitation - Anti-Otx1 + Otx2 antibody (ab21990)

  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) (Mouse monoclonal [SB62a] Anti-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.

  All lanes: Immunoprecipitation - Anti-Otx1 + Otx2 antibody (ab21990)

  Developed using the ECL technique.

  Performed under reducing conditions.

  Exposure time: 90s

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  Western blot - Anti-Otx1 + Otx2 antibody (ab21990)

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

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

  All lanes: OTX1 (Human) - Recombinant Protein at 0.1 µg

  Secondary

  All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) preadsorbed (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

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  This image is courtesy of Erin Bassett

  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Otx1 + Otx2 antibody (ab21990)

  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).

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  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Otx1 + Otx2 antibody (ab21990)

  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.

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  Western blot - Anti-Otx1 + Otx2 antibody (ab21990)

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

  Lane 1: Y79 Lysate at 20 µg

  Lane 2: Y79 Lysate at 20 µg with Otx2 peptide (ab24347)

  Secondary

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

  Predicted band size: 31.6 kDa

  Observed band size: 28 kDa, 37 kDa

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  This image is courtesy of L. Vallier, University of Cambridge

  Immunocytochemistry/ Immunofluorescence - Anti-Otx1 + Otx2 antibody (ab21990)

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

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  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Otx1 + Otx2 antibody (ab21990)
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  Western blot - Anti-Otx1 + Otx2 antibody (ab21990)

  Blocking and diluting buffer and concentration: 5% NFDM/TBST.

  Negative control: HeLa (PMID: 12559959)

  Anti-OTX2 antibody (Anti-OTX2 antibody [RM1205] ab322111, recombinant monoclonal) showed stronger band than Anti-Otx1 + Otx2 antibody (ab21990, polyclonal) with human samples.

  In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (Anti-GAPDH antibody [EPR16891] - Loading Control ab181602) staining at 1/200000 dilution.

  All lanes: Western blot - Anti-OTX2 antibody [RM1205] (Anti-OTX2 antibody [RM1205] ab322111) at 1/1000 dilution

  Lane 1: Y79 (human retinoblastoma cell) whole cell lysate at 20 µg

  Lane 2: HeLa (human cervical adenocarcinoma epithelial cell) whole cell lysate at 20 µg

  Secondary

  All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/100000 dilution

  Observed band size: 32 kDa, 36 kDa

  Exposure time: 180s

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  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Otx1 + Otx2 antibody (ab21990)

  Immunohistochemical analysis of paraffin-embedded Human retina tissue labeling OTX2 with Anti-OTX2 antibody [RM1205] ab322111 at 1/20000 (0.025 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Positive staining on human retina in (A) Anti-OTX2 antibody [RM1205] ab322111 and (B) ab21990. The image on (A) Anti-OTX2 antibody [RM1205] ab322111 is applied with Anti-OTX2 antibody at 1/20000 dilution and (B) ab21990 is applied with Anti-OTX2 antibody at 1/4000 dilution. The section was incubated with Anti-OTX2 antibody [RM1205] ab322111 or ab21990 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Incubate slides with 3% Hydrogen Peroxide for 10 mins at room temperature after secondary antibody incubation to reduce the background. Counterstained with Hematoxylin. Secondary antibody only control: Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins.

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  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Otx1 + Otx2 antibody (ab21990)

  Immunohistochemical analysis of paraffin-embedded Mouse retina tissue labeling OTX2 with Anti-OTX2 antibody [RM1205] ab322111 at 1/20000 (0.025 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Positive staining on mouse retina in (A) Anti-OTX2 antibody [RM1205] ab322111 and (B) ab21990. The image on (A) Anti-OTX2 antibody [RM1205] ab322111 is applied with Anti-OTX2 antibody at 1/20000 dilution and (B) ab21990 is applied with Anti-OTX2 antibody at 1/4000 dilution. The section was incubated with Anti-OTX2 antibody [RM1205] ab322111 or ab21990 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Incubate slides with 3% Hydrogen Peroxide for 10 mins at room temperature after secondary antibody incubation to reduce the background. Counterstained with Hematoxylin. Secondary antibody only control: Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins.