Anti-Optineurin antibody [EPR20654]

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Optineurin antibody [EPR20654] (AB213556)

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.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Optineurin antibody [EPR20654] (AB213556)

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.

• IP

Collaborator

Immunoprecipitation - Anti-Optineurin antibody [EPR20654] (AB213556)

Immunoprecipitation of OPTN in U-2 OS cells. Lysates were prepared and immunoprecipitation was performed using 1.0 μg of ab213556 pre-coupled to prot.A-Sepharose beads. Samples were washed and processed for western blot with ab213556 at 1/5000. SM=10% starting material; UB=10% unbound fraction; IP=immunoprecipitate. These data were provided by YCharOS Inc., an open science company with the mission of characterizing commercially available antibody reagents for all human proteins. Abcam and YCharOS are working together to help address the reproducibility crisis by enabling the life science community to better evaluate commercially available antibodies.

All lanes:

Immunoprecipitation - Anti-Optineurin antibody [EPR20654] (ab213556)

Predicted band size: 66 kDa

false

• IP

Supplier Data

Immunoprecipitation - Anti-Optineurin antibody [EPR20654] (AB213556)

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.

All lanes:

Immunoprecipitation - Anti-Optineurin antibody [EPR20654] (ab213556)

Predicted band size: 66 kDa

false

• IHC-Fr

Supplier Data

Immunohistochemistry (Frozen sections) - Anti-Optineurin antibody [EPR20654] (AB213556)

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).

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Optineurin antibody [EPR20654] (AB213556)

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.

• IHC-Fr

Supplier Data

Immunohistochemistry (Frozen sections) - Anti-Optineurin antibody [EPR20654] (AB213556)

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).

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Optineurin antibody [EPR20654] (AB213556)

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.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Optineurin antibody [EPR20654] (AB213556)

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.

• WB

Lab

Western blot - Anti-Optineurin antibody [EPR20654] (AB213556)

Western blot : Anti-Optineurin antibody [EPR20654] ab213556 staining at 1/500 dilution, shown in green; Mouse anti GAPDH ab8245 loading control staining at 1/20000 dilution, shown in magenta. A band was observed at 73 kDa in Wild-type MCF7 cell lysates with no signal observed at this size in OPTN knockout MCF7 cell line. To generate this image, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3pc Milk in TBS-0.1 % Tween^™ 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit 800CW and Goat anti-Mouse 680RD at 1/20,000 dilution.

All lanes:

Western blot - Anti-Optineurin antibody [EPR20654] (ab213556) at 1/500 dilution

Lane 1:

Wild-type MCF7 whole cell lysate at 20 µg

Lane 2:

OPTN knockout MCF7 whole cell lysate at 20 µg

Lane 3:

U-2 OS whole cell lysate at 20 µg

Lane 4:

Raji whole cell lysate at 20 µg

Secondary

All lanes:

Goat anti-Rabbit 800CW & Goat anti-Mouse 680RD at 1/20000 dilution

Predicted band size: 66 kDa

Observed band size: 73 kDa

false

• WB

Supplier Data

Western blot - Anti-Optineurin antibody [EPR20654] (AB213556)

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).

All lanes:

Western blot - 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:

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

Predicted band size: 66 kDa

Observed band size: 68 kDa

false

• WB

Supplier Data

Western blot - Anti-Optineurin antibody [EPR20654] (AB213556)

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).

All lanes:

Western blot - Anti-Optineurin antibody [EPR20654] (ab213556) at 1/1000 dilution

Lane 1:

Human fetal brain lysate at 10 µg

Lane 2:

Human fetal kidney lysate at 10 µg

Lane 3:

Human placenta lysate at 10 µg

Secondary

All lanes:

Western blot - VeriBlot for IP Detection Reagent (HRP) (<a href='/en-us/products/reagents/veriblot-for-ip-detection-reagent-hrp-ab131366'>ab131366</a>) at 1/4000 dilution

Predicted band size: 66 kDa

Observed band size: 68 kDa

false

• WB

Collaborator

Western blot - Anti-Optineurin antibody [EPR20654] (AB213556)

ab213556 was shown to react with OPTN in wild-type U-2 OS cells in Western blot with loss of signal observed in a OPTN knockout cell line. Wild-type U-2 OS and OPTN knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 5% milk in TBST for 1 hr before incubation with ab213556 overnight at 4 °C at a 1/5000 dilution. Blots were incubated with goat anti-rabbit HRP secondary antibodies at 1/5000 before imaging. These data were provided by YCharOS Inc., an open science company with the mission of characterizing commercially available antibody reagents for all human proteins. Abcam and YCharOS are working together to help address the reproducibility crisis by enabling the life science community to better evaluate commercially available antibodies.

All lanes:

Western blot - Anti-Optineurin antibody [EPR20654] (ab213556) at 1/5000 dilution

Lane 1:

Wild-type U-2 OS cell lysate at 20 µg

Lane 2:

OPTN knockout U-2 OS cell lysate at 20 µg

Predicted band size: 66 kDa

false

• WB

Lab

Western blot - Anti-Optineurin antibody [EPR20654] (AB213556)

ab213556 was shown to react with OPTN in wild-type U-2 OS cells in Western blot with loss of signal observed in a OPTN knockout cell line. Wild-type U-2 OS and OPTN knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 5% milk in TBST for 1 hr before incubation with ab213556 overnight at 4 °C at a 1/500 dilution. Blots were incubated with secondary antibodies at 0.2ug/mL before imaging.

This data was provided by YCharOS Inc., an open science company with the mission of characterizing commercially available antibody reagents for all human proteins. Abcam and YCharOS are working together to help address the reproducibility crisis by enabling the life science community to better evaluate commercially available antibodies.

All lanes:

Western blot - Anti-Optineurin antibody [EPR20654] (ab213556) at 1/500 dilution

Lane 1:

Wild-type U-2 OS cell lysate at 20 µg

Lane 2:

OPTN knockout U-2 OS cell lysate at 20 µg

Observed band size: 66 kDa

false

• WB

Supplier Data

Western blot - Anti-Optineurin antibody [EPR20654] (AB213556)

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).

All lanes:

Western blot - Anti-Optineurin antibody [EPR20654] (ab213556) at 1/1000 dilution

Lane 1:

Rat heart lysate at 10 µg

Lane 2:

Mouse placenta lysate at 10 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

Predicted band size: 66 kDa

Observed band size: 68 kDa

false

• IP

Supplier Data

Immunoprecipitation - Anti-Optineurin antibody [EPR20654] (AB213556)

Immunoprecipitation of OPTN in c-43 cells. Lysates were prepared and immunoprecipitation was performed using 1.0 μg of ab213556 pre-coupled to prot.A-Sepharose beads. Samples were washed and processed for western blot with ab213556 at 1/5000. This data was kindly provided by the YCharOS Inc., an open science company with the mission of characterizing every commercially available antibody reagent. Abcam are working with YCharOS to support their mission of antibody characterisation using knock out cell lines.

All lanes:

Immunoprecipitation - Anti-Optineurin antibody [EPR20654] (ab213556)

Predicted band size: 66 kDa

false