ab213556 staining Optineurin in Mouse retina tissue sections by Immunohistochemistry (IHC-Fr - frozen sections). Tissue was fixed with 4% paraformaldehyde, permeabilized with 0.2% Triton. Samples were incubated with primary antibody (1/100). An Alexa Fluor^® 488 Goat anti-Rabbit secondary (1/1000) was used as the secondary antibody. Counter stained with DAPI.

Heat mediated antigen retrieval using sodium citrate buffer (10mM citrate pH 6.0 + 0.05% Tween-20)

Positive staining on mouse retina (PMID: 15607428).

All lanes : Anti-Optineurin antibody [EPR20654] (ab213556) at 1/1000 dilution

Lane 1 : Human skeletal muscle lysate at 20 µg
Lane 2 : 293T (Human epithelial cell line from embryonic kidney) whole cell lysate at 20 µg
Lane 3 : Rat brain lysate at 20 µg
Lane 4 : Mouse retina lysate at 20 µg
Lane 5 : Rat retina lysate at 20 µg
Lane 6 : Rat placenta lysate at 20 µg
Lane 7 : NIH/3T3 (Mouse embryonic fibroblast cell line) whole cell lysate at 10 µg

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

Predicted band size: 66 kDa
Observed band size: 68 kDa why is the actual band size different from the predicted?

Blocking/Dilution buffer: 5% NFDM/TBST.

Exposure time: Lane 1-3/7: 3 minutes; Lane 4-6: 10 seconds.

The MW observed is consistent with the literature (PMID 20174559, PMID 24983867, PMID 11834836).

Immunohistochemical analysis of paraffin-embedded human retina tissue labeling Optineurin with ab213556 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) Ready to use. Cytoplasmic staining on human retina (PMID: 15607428).

Counter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) Ready to use.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

ab213556 staining Optineurin in the NIH/3T3 (mouse embryonic fibroblast) by ICC/IF (Immunocytochemistry/immunofluorescence). Cells were fixed with 4% Paraformaldehyde, permeabilized with 0.1% Triton X-100. Samples were incubated with primary antibody (1/50). ab150077 Alexa Fluor^® 488 Goat anti-Rabbit secondary (1/1000) was used as the secondary antibody. Ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) (1/200) was used as a counterstain. 

ab213556 staining Optineurin in Rat retina tissue sections by Immunohistochemistry (IHC-Fr - frozen sections). Tissue was fixed with 4% paraformaldehyde, permeabilized with 0.2% Triton. Samples were incubated with primary antibody (1/100). An Alexa Fluor^® 488 Goat anti-Rabbit secondary (1/1000) was used as the secondary antibody. Counter stained with DAPI.

Heat mediated antigen retrieval using sodium citrate buffer (10mM citrate pH 6.0 + 0.05% Tween-20)

Positive staining on rat retina (PMID: 15607428).

ab213556 staining Optineurin in the HeLa (human cervix adenocarcinoma epithelial cell) by ICC/IF (Immunocytochemistry/immunofluorescence). Cells were fixed with 4% Paraformaldehyde, permeabilized with 0.1% Triton X-100. Samples were incubated with primary antibody (1/50). ab150077 Alexa Fluor^® 488 Goat anti-Rabbit secondary (1/1000) was used as the secondary antibody. Ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) (1/200) was used as a counterstain. 

All lanes : Anti-Optineurin antibody [EPR20654] (ab213556) at 1/1000 dilution

Lane 1 : Human fetal brain lysate
Lane 2 : Human fetal kidney lysate
Lane 3 : Human placenta lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : VeriBlot for IP Detection Reagent (HRP) (ab131366) at 1/4000 dilution

Predicted band size: 66 kDa
Observed band size: 68 kDa why is the actual band size different from the predicted?

Blocking/Dilution buffer: 5% NFDM/TBST.

Exposure time: Lane 1: 3 seconds; Lane 2/3: 30 seconds.

The MW observed is consistent with the literature (PMID 20174559, PMID 24983867, PMID 11834836).

Immunohistochemical analysis of paraffin-embedded human cerebrum tissue labeling Optineurin with ab213556 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) Ready to use. Cytoplasmic staining on human cerebrum (PMID: 26303227).

Counter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) Ready to use.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

All lanes : Anti-Optineurin antibody [EPR20654] (ab213556) at 1/1000 dilution

Lane 1 : Rat heart lysate
Lane 2 : Mouse placenta lysate

Lysates/proteins at 10 µg per lane.

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

Predicted band size: 66 kDa
Observed band size: 68 kDa why is the actual band size different from the predicted?

Blocking/Dilution buffer: 5% NFDM/TBST.

Exposure time: Lane 1: 3 minutes; Lane 2: 10 seconds.

The MW observed is consistent with the literature (PMID 20174559, PMID 24983867, PMID 11834836).

Immunohistochemical analysis of paraffin-embedded mouse retina tissue labeling Optineurin with ab213556 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) Ready to use. Cytoplasmic staining on mouse retina (PMID: 15607428).

Counter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) Ready to use.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Immunohistochemical analysis of paraffin-embedded mouse cerebrum tissue labeling Optineurin with ab213556 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) Ready to use. Cytoplasmic staining on mouse cerebrum (PMID: 26303227).

Counter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) Ready to use.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Immunohistochemical analysis of paraffin-embedded rat cerebellum tissue labeling Optineurin with ab213556 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) Ready to use. Cytoplasmic staining on rat cerebellum (PMID: 26303227).

Counter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) Ready to use.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Optineurin was immunoprecipitated from 0.35 mg of 293T (Human epithelial cell line from embryonic kidney) whole cell lysate with ab213556 at 1/40 dilution. Western blot was performed from the immunoprecipitate using ab213556 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/10,000 dilution

Lane 1: 293T whole cell lysate 10 μg (Input).

Lane 2: ab213556 IP in 293T whole cell lysate.

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab213556 in 293T whole cell lysate.

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

Exposure time: 3 minutes.