Anti-Optineurin antibody [EPR23059-124]

• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Anti-Optineurin antibody [EPR23059-124] (AB242146)

Intracellular flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized HeLa (human cervix adenocarcinoma) cells labelling Optineurin with ab242146 at 1/50 (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). A Goat anti rabbit IgG (Alexa Fluor^®488, ab150077) at 1/2000 dilution was used as the secondary antibody.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-Optineurin antibody [EPR23059-124] (AB242146)

ab242146 was shown to react with OPTN in wild-type U20S cells in immunocytochemistry with loss of signal observed in a OPTN knockout cell line. Wild-type and knockout cells were mixed and pelleted at a 1 : 1 ratio on coverslips. The cells were fixed with 4% paraformaldehyde (15 min) then permeabilized with 0.1% Triton X-100 (10min) and then blocked with 1x PBS, 0.01% Triton X-100, 5% BSA, 5% NGS. The cells were then incubated with ab242146 at dilution overnight at 4°C followed by a further incubation at room temperature for 1h with a goat anti-rabbit secondary antibody to (Alexa Fluor® 555) at 0.5 µg/ml. Acquisition of the green (wild-type), red (antibody staining) and far-red (knockout) channels was performed. Representative grayscale images of the red channel are shown. Wild-type and knockout cells are outlined with yellow and magenta dashed line, respectively. Schematic representation of the mosaic strategy used is shown on the bottom-right panel. Image was acquired with a Zeiss(LSM-880).

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.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Optineurin antibody [EPR23059-124] (AB242146)

Immunohistochemical analysis of paraffin-embedded Human cerebrum tissue labeling Optineurin with ab242146 at 1/500 dilution (0.98 ug/ml) followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Cytoplasmic staining on human cerebrum. The section was incubated with ab242146 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND®RX instrument. Counterstained with Hematoxylin.

Secondary antibody only control : Secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20mins.

• IP

Lab

Immunoprecipitation - Anti-Optineurin antibody [EPR23059-124] (AB242146)

Immunoprecipitation of OPTN in U20S cells. Lysates were prepared and immunoprecipitation was performed using 1.0 µg of ab242146 pre-coupled to Protein A beads. Samples were washed and processed for western blot with OPTN Monoclonal Antibody at 1/10000

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.

Lane 1:

Immunoprecipitation at 1/10000 dilution

Lane 3:

Immunoprecipitation - Anti-Optineurin antibody [EPR23059-124] (ab242146) at 1 µg

All lanes:

U20S cells

Observed band size: 66 kDa

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• WB

Lab

Western blot - Anti-Optineurin antibody [EPR23059-124] (AB242146)

Western blot : Anti-Optineurin antibody [EPR23059-124] ab242146 staining at 1/1000 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 [EPR23059-124] (ab242146) at 1/1000 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

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• WB

Lab

Western blot - Anti-Optineurin antibody [EPR23059-124] (AB242146)

ab242146 was shown to react with OPTN in wild-type U-2 OS cells in Western blot with loss of signal observed in OPTN knockout cell line ab261906. 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 ab242146 overnight at 4 °C at a 1/1000 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 [EPR23059-124] (ab242146) at 1/1000 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

Lab

Western blot - Anti-Optineurin antibody [EPR23059-124] (AB242146)

Blocking and diluting buffer and concentration : 5% NFDM/TBST The molecular weight observed is consistent with what has been described in the literature (PMID : 27620379).

Exposure time : Lane 1 : 15 seconds Lane 2 : 70 seconds

All lanes:

Western blot - Anti-Optineurin antibody [EPR23059-124] (ab242146) at 1/1000 dilution

Lane 1:

Human brain lysate at 20 µg

Lane 2:

Hela (human cervix adenocarcinoma epithelial cell) 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

Predicted band size: 66 kDa

Observed band size: 74 kDa

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