- 711879 - Invitrogen Antibodies OPTN antibody

Submitted references Identification of novel lipid droplet factors that regulate lipophagy and cholesterol efflux in macrophage foam cells.

Robichaud S, Fairman G, Vijithakumar V, Mak E, Cook DP, Pelletier AR, Huard S, Vanderhyden BC, Figeys D, Lavallée-Adam M, Baetz K, Ouimet M
Autophagy 2021 Nov;17(11):3671-3689

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Antibody specificity was demonstrated by detection of differential basal expression of the target across tissue models owing to their inherent genetic constitution. Higher expression of Optineurin was observed specifically in Mouse Eye in comparison to Mouse Liver using Anti-Optineurin Recombinant Rabbit Polyclonal Antibody (Product # 711879) in Western blot.

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 Oligoclonal Antibody (Product # 711879, 1:1000 dilution) and Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 0.25 µg/ml 1:4000 dilution). Loss of signal upon CRISPR mediated knockout (KO) confirms that antibody is specific to Optineurin.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Knockdown of Optineurin was achieved by transfecting NIH/3T3 cells with Optineurin specific siRNA (Silencer® select Product # s19720+s19718). Western blot analysis (Fig a) was performed using whole cell extracts from Optineurin knockdown cells (Lane 3), non-specific scrambled siRNA transfected cells (Lane 2) and untransfected cells (Lane 1). The blots were probed with Anti-Optineurin Recombinant Rabbit Polyclonal Antibody (Product # 711879, 1-3 µg/mL) and Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 0.25 µg/mL, 1:4000 dilution). Densitometric analysis of this Western blot is shown in histogram (Fig b). Loss of signal upon siRNA mediated knock down confirms that antibody is specific to Optineurin.

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) and Mouse Eye (Lane 7). The blots were probed with Anti-Optineurin Recombinant Rabbit Polyclonal Antibody (Product # 711879, 2.5 µg/mL) and detected by chemiluminescence using Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 0.25 µg/mL, 1:4000 dilution). A 66 kDa band corresponding to Optineurin was observed across the cell lines and relevant tissues tested. Known quantity of protein samples were electrophoresed using Novex®NuPAGE®4-12% Bis-Tris gel (Product # NP0322BOX), XCell SureLock™ Electrophoresis System (Product # EI0002) and Novex® Sharp Pre-Stained Protein Standard (Product # LC5800). Resolved proteins were then transferred onto a nitrocellulose membrane with iBlot® Dry Blotting System (Product # IB21001). The membrane was probed with the relevant primary and secondary Antibody following blocking with 5% skimmed milk. Chemiluminescent detection was performed using Pierce™ ECL Western blotting Substrate (Product # 32106).

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 polyclonal antibody (Product # 711879) at a dilution of 1:5,000 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)
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 polyclonal antibody (Product # 711879) 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 Pierce ECL Western Blotting Substrate (Product # 32106). 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)
Main image
Experimental details: For immunofluorescence analysis, NIH 3T3 cells were fixed and permeabilized for detection of endogenous Optineurin using Anti- Optineurin Recombinant Rabbit Polyclonal Antibody (Product # 711879, 5 µg/mL) and labeled with Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A27034, 1:2000). 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)
Main image
Experimental details: Immunofluorescence of OPTN was performed using U2OS wild-type and OPTN KO cells that were transfected with a green or a deep red fluorescent dye, respectively. Post-transfection, WT and KO cells were mixed and plated to a 1:1 ratio on coverslips as a mosaic and incubated for 24 hrs. Cells were fixed in 4% PFA (in PBS) for 15 min; cells were permeabilized with 0.1% Triton X-100 for 10 min at RT and blocked with PBS with 5% BSA, 5% goat serum, and 0.01% Triton X-100 for 30 min. Cells were stained with the OPTN recombinant polyclonal antibody (Product # 711879) at a 1:500 dilution overnight at 4°C. Secondary antibody incubation was performed using 1 µg/mL of Goat anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 555 antibody (Product # A21429) together with DAPI for 1 hr. Imaging was performed with a 40X oil objective and analysis was performed using Image J. Cell image represents a single focal plane; WT and KO cells are outlined with a yellow (WT) or magenta (KO) dashed line. 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)
Main image
Experimental details: Immunofluorescence analysis of mouse Retina tissue: Frozen sections were fixed with 4% PFA for 20 min, permeabilized using 0.1% Triton-X 100 for 10 mins and blocked for 1 hour with 2% BSA. Transverse sections of mouse Retina were incubated with Anti-Optineurin Recombinant Rabbit Polyclonal Antibody (Product # 711879, 1:250 dilution) overnight at 4°C, followed by Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A27034, 1:2000, 45 mins). Nuclei (blue) were stained using SlowFade® Gold Antifade Mountant with DAPI (Product # S36938), Panel a) represents staining with the matched isotype control. Panel b) shows a representative mouse retinal section stained for Optineurin (green). The images were captured at 20X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 4. Selective autophagy receptors (SARs) OPTN, NBR1 and SQSTM1 selectively localize to foam cell lipid droplets (LDs). (A-D) Immunostaining for OPTN (A), SQSTM1 (B), NBR1 (C) or CALCOCO2 (D) in agLDL-loaded mouse bone marrow-derived macrophage foam cells stained with BODIPY 493/503 to label LD neutral lipids, with areas of interest circled. At right, quantification of the percent of cellular LDs tagged with SARs in chloroquine-treated cells (CQ) as compared to control (Ctrl) is shown. Data are expressed as fold-change for the chloroquine treatment relative to untreated from one experiment representative of 3 independent experiments with similar results (mean +- s.e.m). ** P < 0.005, *** P < 0.0005. Representative images are from CQ-treated cells. Scale bar: 5 mum

711879

Antibody data