Anti-Otx2 antibody [EPR3348]

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-Otx2 antibody [EPR3348] (AB92326)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized Y79 (human retinoblastoma cell) cells labelling Otx2 with ab92326 at 1/500 (2.098 µg/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) preadsorbed antibody at 1/1000 (2 µg/ml) dilution (Green).

Confocal image showing nuclear staining in Y79 cell line (shown in green). The counterstain was observed in magenta. Nuclear DNA was labeled with DAPI (shown in blue).
Negative control : HeLa (PMID : 12559959).
Image was taken with a confocal microscope(Leica-Microsystems, TCS SP8).

ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor^® 594) was used to counterstain tubulin at 1/200 (2.5 µg/ml) dilution (Magenta).

Secondary antibody only control : Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) preadsorbed at 1/1000 (2 µg/ml) dilution.

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-Otx2 antibody [EPR3348] (AB92326)

Flow cytometric analysis of 4% paraformaldehyde fixed 0.1% Tween-20l permeabilized HeLa (human cervical adenocarcinoma epithelial cell, Left) / Y79(human retinoblastoma cell, Right) cells labelling Otx2 with ab92326 at 1/1000 dilution (0.01µg) (Red) compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue).

Goat Anti-Rabbit IgG (Alexa Fluor^® 488, ab150081) at 1/5000 dilution was used as the secondary antibody.

Negative control : HeLa (PMID : 12559959).

• WB

Lab

Western blot - Anti-Otx2 antibody [EPR3348] (AB92326)

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

Lanes 1 - 2:

Western blot - Anti-Otx2 antibody [EPR20375] (<a href='/en-us/products/primary-antibodies/otx2-antibody-epr20375-ab183951'>ab183951</a>) at 1/1000 dilution

Lanes 3 - 4:

Western blot - Anti-Otx1 antibody [EPR3347] (<a href='/en-us/products/primary-antibodies/otx1-antibody-epr3347-ab92515'>ab92515</a>) at 1/1000 dilution

Lanes 5 - 6:

Western blot - Anti-Otx2 antibody [EPR3348] (ab92326) at 1/1000 dilution

Lanes 1, 3 and 5:

GST-tagged Human OTX1 full length recombinant protein at 10 ng

Lanes 2, 4 and 6:

His-tagged Human OTX2 full length recombinant protein at 10 ng

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Observed band size: 35 kDa,75 kDa

false

• WB

Lab

Western blot - Anti-Otx2 antibody [EPR3348] (AB92326)

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

All lanes:

Western blot - Anti-Otx2 antibody [EPR3348] (ab92326) at 1/1000 dilution

All lanes:

Y79 cell lysate at 10 µg

Secondary

All lanes:

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

Predicted band size: 32 kDa

Observed band size: 32 kDa

false

• WB

Lab

Western blot - Anti-Otx2 antibody [EPR3348] (AB92326)

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

ab129002 was used as a Vinculin loading control at a 1/10000 dilution.

Negative control : spleen
The membrane was incubated with ab92326 at 4° overnight.

All lanes:

Western blot - Anti-Otx2 antibody [EPR3348] (ab92326) at 1/1000 dilution

Lane 1:

Mouse retina tissue lysate at 20 µg

Lane 2:

Mouse spleen tissue lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Observed band size: 36 kDa

false

Exposure time: 70s

• WB

Lab

Western blot - Anti-Otx2 antibody [EPR3348] (AB92326)

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

ab129002 was used as a Vinculin loading control at a 1/10000 dilution.

Negative control : spleen

All lanes:

Western blot - Anti-Otx2 antibody [EPR3348] (ab92326) at 1/1000 dilution

Lane 1:

Rat retina tissue lysate at 20 µg

Lane 2:

Rat spleen tissue lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Observed band size: 32 kDa

false

Exposure time: 147s

• WB

Unknown

Western blot - Anti-Otx2 antibody [EPR3348] (AB92326)

All lanes:

Western blot - Anti-Otx2 antibody [EPR3348] (ab92326) at 1/1000 dilution

All lanes:

Y79 cell lysate at 10 µg

Secondary

All lanes:

HRP labelled goat anti-rabbit IgG at 1/1000 dilution

Predicted band size: 32 kDa

false