- 702766 - Invitrogen Antibodies OPTN antibody

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis was performed on whole cell extracts (30 µg lysate) of NIH/3T3 (Lane 1), NCCIT (Lane 2), HeLa (Lane 3) and tissue extracts of Mouse Brain (Lane 4), Rat Brain (Lane 5), Mouse Liver (Lane 6), Mouse Eye (Lane 7) and Rat Eye (Lane 8). The blots were probed with Anti-Optineurin Recombinant Rabbit Monoclonal Antibody (Product # 702766, 1:200 dilution) and detected by chemiluminescence using Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 0.25 µg/mL, 1:4,000 dilution). A 66 kDa band corresponding to Optineurin was observed across the cell lines and relevant tissues tested.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis was performed on Tissue extracts (30 µg lysate) of Mouse Kidney (Lane 1) Mouse Liver (Lane 2) and Mouse Eye (Lane 3). The blots were probed with Anti-Optineurin Recombinant Rabbit Monoclonal Antibody (Product # 702766, 1:200 dilution) and detected by chemiluminescence using Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 0.25 µg/mL, 1:4,000 dilution). A 66 kDa band corresponding to Optineurin was observed across the relevant tissues tested.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot of OPTN was performed by loading 30 µg of WT (lane 1) and OPTN CRISPR KO (lane 2) U2OS cell lysates in RIPA buffer onto a 5-16% gradient polyacrylamide gel. Proteins on the blots were visualized with Ponceau staining (below immunoblot). Proteins were transferred to nitrocellulose membrane and blocked in 5% milk for 1 hr. OPTN was detected at approximately 66 kDa using an OPTN recombinant monoclonal antibody (Product # 702766) at a dilution of 1:200 in 5% BSA in TBS with 0.1% Tween 20 (TBST) overnight at 4°C. The peroxidase-conjugated secondary antibody (Product # 65-6120) was diluted to 0.2 µg/mL in TBST with 5% milk for 1 hr. Chemiluminescent detection was performed using Pierce ECL Western Blotting Substrate (Product # 32106) or with SuperSignal West Femto (Product # 34096). Data courtesy of YCharOS Inc., an open science company with the mission of characterizing commercially available antibodies using knockout validation.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis of Optineurin was performed by loading 30 µg of HCT 116 control (Lane 1), HCT 116 Optineurin knockout (Lane 2) whole cell lysates. The blot was probed with ABfinity™ Anti-Optineurin Recombinant Rabbit Monoclonal Antibody (Product # 702766, 1:200 dilution) and Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 1:4,000 dilution). Loss of signal upon CRISPR mediated knockout (KO) confirms that antibody is specific to Optineurin.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot analysis of Optineurin was performed by loading 10 µg of WT (lane 1) and OPTN CRISPR KO (lane 2) U2OS cell lysates in RIPA buffer onto a 4-15% gradient polyacrylamide gel. Proteins were transferred to nitrocellulose membrane and blocked in 5% milk. Ponceau stained transfer of blot is shown. Optineurin was detected at approximately 70 kDa using an Optineurin recombinant monoclonal antibody (Product # 702766) at a dilution of 1:5,000 in 5% BSA in TBST overnight at 4 deg, followed by secondary antibody diluted to 0.2 µg/mL using Goat anti-Rabbit IgG (H+L) HRP antibody (Product # 65-6120). Chemiluminescent detection was performed using SuperSignalWest Femto Maximum Sensitivity Substrate (Product # 34095). Longer exposure is shown in the smaller cropped panel. Data courtesy of YCharOS Inc., an open science company with the mission of characterizing commercially available antibodies using knockout validation.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: For immunofluorescence analysis, HeLa cells were fixed and permeabilized for detection of endogenous Optineurin using Anti- Optineurin Recombinant Rabbit Monoclonal Antibody (Product # 702766, 1:100 dilution) and labeled with Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A27034, 1:2,000). Panel a) shows representative cells that were stained for detection and localization of Optineurin protein (green), Panel b) is stained for nuclei (blue) using SlowFade® Gold Antifade Mountant with DAPI (Product # S36938). Panel c) represents cytoskeletal F-actin staining using Rhodamine Phalloidin (Product # R415, 1:300). Panel d) is a composite image of Panels a, b and c clearly demonstrating cytoplasmic localization of Optineurin. Panel e) represents control cells without primary antibody to assess background. The images were captured at 60X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: For immunofluorescence analysis, iPSC differentiated to Retinal Ganglion cells were fixed and permeabilized for detection of endogenous Optineurin using anti-Optineurin Recombinant Rabbit monoclonal Antibody (Product # 702766, 1:100 dilution) and labeled with Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A27034, 1:2,000). Panel a) shows undifferentiated iPSC cells, Panel b) is the image of iPSC differentiated to Retinal ganglion cells showing cytoplasmic localization of Optineurin.

702766