CD274 Antibody

• Catalog Number: orb1239835
• Category: Antibodies
• Description: CD274 Antibody
• Species/Host: Rabbit
• Clonality: Polyclonal
• Tested applications: ELISA, FC, IF, IHC-P, WB
• Reactivity: Human, Mouse, Rat
• Isotype: IgG
• Immunogen: Anti-PD-L1 antibody (orb1239835) was raised against a peptide corresponding to 17 amino acids near the center of human PD-L1 isoform 1. The immunogen is located within amino acids 60 - 110 of PD-L1.
• Concentration: 1 mg/mL
• Dilution range: WB: 1-2 μg/mL; IHC-P: 2.5-5 μg/mL; IF: 20 μg/mL; Flow Cyt: 0.5 μg/mL. Antibody validated: Western Blot in human and mouse samples; Immunohistochemistry in human and rat samples; Immunofluorescence in human and rat samples and Flow Cytometry in mouse samples. All other applications and species not yet tested.
• Form/Appearance: Liquid
• Conjugation: Unconjugated
• MW: Predicted: 33 kDa Observed: 37 kDa
• Target: CD274
• UniProt ID: Q9NZQ7
• NCBI: NP_054862
• Storage: PD-L1 antibody can be stored at 4°C for three months and -20°C, stable for up to one year. As with all antibodies care should be taken to avoid repeated freeze thaw cycles. Antibodies should not be exposed to prolonged high temperatures.
• Buffer/Preservatives: PD-L1 Antibody is supplied in PBS containing 0.02% sodium azide.
• Alternative names: PD-L1 Antibody: B7-H, B7H1, PDL1, PD-L1, PDCD1L1,
• Note: For research use only
• Expiration Date: 12 months from date of receipt.

Western Blot Validation of PD-L1 in HeLa Cells. Loading: 15 µg of lysates per lane. Antibodies: orb1239835 (A: 1 µg/mL, B: 2 µg/mL), 1 h incubation at RT in 5% NFDM/TBST. Secondary: Goat anti-rabbit IgG HRP conjugate at 1:10000 dilution.

Independent Antibody Validation (IAV) via Protein Expression Profile in Human and Mouse cell lines. Loading: 15 µg of lysates per lane. Antibodies: orb1239835 (2 µg/mL), orb1239770 (2 µg/mL), and beta-actin (1 µg/mL), 1 h incubation at RT in 5% NFDM/TBST. Secondary: Goat anti-rabbit and or anti-mouse IgG HRP conjugate at 1:10000 and 1:5000 dilution, respectively.

Validation with PD-L1 siRNA Knockdown in HeLa Cells. HeLa cells were transfected with control siRNAs (lane 1) or PD-L1 siRNAs (lane 2) Loading: 10 µg of HeLa whole cell lysates per lane. Antibodies: orb1239835 (2 µg/mL) and GAPDH (orb1239765, 0.02 µg/mL), 1 h incubation at RT in 5% NFDM/TBST. Secondary: Goat anti-mouse IgG HRP conjugate at 1:5000 dilution.

Validation with PD-L1 overexpression in 293 cells. Loading: Lysates/proteins at 15 µg per lane. Lane 1: non-transfected 293 cells, Lane 2: PD-L1 overexpressed 293 cells. Antibodies: orb1239835 (1 µg/mL). 1 h incubation at RT in 5% NFDM/TBST. Secondary: Goat anti-rabbit IgG HRP conjugate at 1:10000 dilution.

Immunohistochemistry Validation of PD-L1 in Human Tonsil Cells. Immunohistochemical analysis of paraffin-embedded human tonsil tissue using anti-PD-L1 antibody (orb1239835) at 5 µg/ml. Tissue was fixed with formaldehyde and blocked with 10% serum for 1 h at RT; antigen retrieval was by heat mediation with a citrate buffer (pH6). Samples were incubated with primary antibody overnight at 4°C. A goat anti-rabbit IgG H&L (HRP) at 1/250 was used as secondary. Counter stained with Hematoxylin.

Immunofluorescence Validation of PD-L1 in Human Heart. Immunofluorescent analysis of 4% paraformaldehyde-fixed human heart tissue labeling PD-L1 with orb1239835 at 20 µg/mL, followed by goat anti-rabbit IgG secondary antibody at 1/500 dilution (red). Image showing both membrane and cytoplasmic staining on human heart tissue.

Flow Cytometry Validation of PD-L1. Overlay histogram showing A-20 cells stained with orb1239835 (red line, 1µg/1x10 6 cells). 1 h incubation at 4°C in 2% FBS/PBS. Followed by secondary antibody 488 goat anti-rabbit IgG (H+L) at 1/500 dilution for 1 h 4°C. Isotype control antibody (Green line) was mouse IgG1 (1µg/1x10 6 cells) used under the same conditions. Acquisition of > 10000 events was performed.

Immunohistochemistry Validation of PD-L1 in Rat Heart. Immunohistochemical analysis of paraffin-embedded rat heart tissue using anti-PD-L1 antibody (orb1239835) at 5 µg/ml. Tissue was fixed with formaldehyde and blocked with 10% serum for 1 h at RT; antigen retrieval was by heat mediation with a citrate buffer (pH6). Samples were incubated with primary antibody overnight at 4°C. A goat anti-rabbit IgG H&L (HRP) at 1/250 was used as secondary. Counter stained with Hematoxylin.

Immunohistochemistry Validation of PD-L1 in Human Heart. Immunohistochemical analysis of paraffin-embedded human heart tissue using anti-PD-L1 antibody (orb1239835). Tissue was fixed with formaldehyde and blocked with 10% serum for 1 h at RT; antigen retrieval was by heat mediation with a citrate buffer (pH6). Samples were incubated with primary antibody overnight at 4°C. A goat anti-rabbit IgG H&L (HRP) at 1/250 was used as secondary. Counter stained with Hematoxylin.

Immunofluorescence Validation of PD-L1 in Rat Heart. Immunofluorescence analysis of 4% paraformaldehyde-fixed rat heart tissue labeling PD-L1 with orb1239835 at 20 µg/ml, followed by goat anti-rabbit IgG secondary antibody at 1/250 dilution (red).

Immunohistochemistry Validation of PD-L1 in Human Tumors (Gadiot et al., 2011). Immunohistochemical analysis of patient tumors labeling PD-L1 with anti-PD-L1 antibodies (orb1239835). Several anti-PD-L1 antibodies were tested for staining.

Immunohistochemistry Validation of PD-L1 in Human thyroid cancer (Angell et al., 2014). Immunohistochemical analysis of PD-L1 expression in human thyroid cancer with anti-PD-L1 antibodies (orb1239835). Placenta was used a positive control.

Immunohistochemistry Validation of PD-L1 in Human Lung Cancer (Ilie et al., 2015). Surgical specimens (left panel) and matched biopsy specimens (right panel). PD-L1-positive (A, B) and PD-L1-negative (C, D) tumors.

Immunohistochemistry Validation of PD-L1 in Human Lung adenocarcinoma (Heymann et al., 2017). HE staining (left panel) and PD-L1 expression (right panel) in the tumor and pleural fluid for a patient with lung adenocarcinoma. PD-L1 expression detected by anti-PD-L1 antibodies (orb1239835) demonstrated membranous staining in approximately 80% of tumor cells (C) and in approximately 75% of tumor cells (D), respectively.