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<strong>Figure 1 Western Blot Validation of PD-L1 in HeLa Cells</strong><br>Loading: 15 μg of lysates per lane.Antibodies: 4059 (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.
<strong>Figure 2 Independent Antibody Validation (IAV) via Protein Expression Profile in Human and Mouse cell lines</strong><br>Loading: 15 μg of lysates per lane.Antibodies: 4059 (2 μg/mL), RF16035 (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.
<strong>Figure 3 Validation with PD-L1 siRNA Knockdown in HeLa Cells</strong><br> 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: 4059 (2 μg/mL) and GAPDH (3783, 0.02 μg/mL), 1 h incubation at RT in 5% NFDM/TBST.Secondary: Goat anti-mouse IgG HRP conjugate at 1:5000 dilution.
<strong>Figure 4 Validation with PD-L1 overexpression in 293 cells </strong><br> Loading: Lysates/proteins at 15 &#956g per lane.Lane 1: non-transfected 293 cellsLane 2: PD-L1 overexpressed 293 cellsAntibodies: 4059 (1 &#956g/mL). 1 h incubation at RT in 5% NFDM/TBST.Secondary: Goat anti-rabbit IgG HRP conjugate at 1:10000 dilution.
<strong>Figure 5 Immunohistochemistry Validation of PD-L1 in Human Tonsil Cells</strong><br> Immunohistochemical analysis of paraffin-embedded human tonsil tissue using anti-PD-L1 antibody (4059) 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.
<strong>Figure 6 Immunofluorescence Validation of PD-L1 in Human Heart </strong><br>Immunofluorescent analysis of 4% paraformaldehyde-fixed human heart tissue labeling PD-L1 with 4059 at 20 &#956g/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.
<strong>Figure 7 Flow Cytometry Validation of PD-L1 </strong><br>Overlay histogram showing A-20 cells stained with 4059 (red line, 1μg/1x10<sup>6</sup> 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<sup>6</sup> cells) used under the same conditions. Acquisition of >10,000 events was performed.
<strong>Figure 8 Immunohistochemistry Validation of PD-L1 in Rat Heart</strong><br>Immunohistochemical analysis of paraffin-embedded rat heart tissue using anti-PD-L1 antibody (4059) 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.
<strong>Figure 9 Immunohistochemistry Validation of PD-L1 in Human Heart</strong><br>Immunohistochemical analysis of paraffin-embedded human heart tissue using anti-PD-L1 antibody (4059) at 2.5 °g/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.
<strong>Figure 10 Immunofluorescence Validation of PD-L1 in Rat Heart</strong><br>Immunofluorescence analysis of 4% paraformaldehyde-fixed rat heart tissue labeling PD-L1 with 4059 at 20 μg/ml, followed by goat anti-rabbit IgG secondary antibody at 1/250 dilution (red).
<strong>Figure 11 Immunofluorescence Validation of PD-L1 in tumors in Human Cells (Dhar et al., 2018) </strong><br>(A) Several antibody brands were first tested with RBCs, WBCs, and HeLa cells: BioLegend, ProSci, and eBioscience. <strong>ProSci (4059) was chosen as it provided the highest staining intensity</strong><br>. (B, C) Using the optimal conditions of anti-PDL1 (ProSci Inc) at a concentration of 50μg/mL, following by goat anti-rabbit Alexa Fluor 647, PDL-1 staining was tested on several lung cancer cell lines: A549 (adenocarcinoma), H1703 (adenocarcinoma), H3255 (squamous) and WBCs as a control. (D) Once validated, patient samples were stained for PD-L1, CK, CD45, DAPI.
<strong>Figure 12 Immunohistochemistry Validation of PD-L1 in Human Tumors (Gadiot et al., 2011) </strong><br>Immunohistochemical analysis of patient tumors labeling PD-L1 with anti-PD-L1 antibodies (4059). Several anti-PD-L1 antibodies were tested for staining, <strong> “Only 1 antibody gave no background staining and was competitively blocked by the addition of PD-L1Fc protein (ProSci, #4059)”. <strong>
<strong>Figure 13 Immunohistochemistry Validation of PD-L1 in Human thyroid cancer (Angell et al., 2014) </strong><br>Immunohistochemical analysis of PD-L1 expression in human thyroid cancer with anti-PD-L1 antibodeis (4059). Placenta was used a positive control.
<strong>Figure 14 Immunohistochemistry Validation of PD-L1 in Human Lung Cancer (Ilie et al., 2015) </strong><br>Surgical specimens (left panel) and matched biopsy specimens (right panel). PD-L1-positive (A,B) and PD-L1-negative (C,D) tumors.
<strong>Figure 15 Immunohistochemistry Validation of PD-L1 in Human Lung adenocarcinoma (Heymann et al., 2017) </strong><br> 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 (4059) demonstrated membranous staining in approximately 80% of tumor cells (C) and in approximately 75% of tumor cells (D), respectively.
Figure 1 Western Blot Validation of PD-L1 in HeLa Cells
Loading: 15 μg of lysates per lane.Antibodies: 4059 (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.
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PD-L1 Antibody

CATALOG NUMBER: 4059

Clonality:
Polyclonal
Tested Applications:
ELISA, Flow, IF, IHC-P, WB
Host Species:
Rabbit
Species Reactivity:
Human, Mouse, Rat
Conjugate:
Unconjugated
Specifications
Host Species:
Rabbit
Species Reactivity:
Human, Mouse, Rat
Homology:
Predicted species reactivity based on immunogen sequence: Rat: (77%), Mouse: (71%)
Immunogen:
Anti-PD-L1 antibody (4059) 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.
Conjugate:
Unconjugated
Tested Applications:
ELISA, Flow, IF, IHC-P, WB
Application Note:
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.
Specificity:
PD-L1 antibody has no cross-reactivity to PD-L2.
Positive Control 1:
Cat. No. 1201 - HeLa Cell Lysate
Positive Control 2:
Cat. No. 1224 - Daudi Cell Lysate
Positive Control 3:
Cat. No. 1204 - K562 Cell Lysate
Positive Control 4:
Cat. No. 10-501 - Human Heart Tissue Slide
Positive Control 5:
Cat. No. 1301 - Human Heart Tissue Lysate
Predicted Molecular Weight:
Predicted: 33 kDa
Observed: 37 kDa
Validation:

Independent Antibody Validation (Figure 2) shows similar PD-L1 expression profile in both human and mouse cell lines detected by two independent anti-PD-L1 antibodies that recognize different epitopes, 4059 against the center of human PD-L1 and RF16035 against the extracellular domain.  PD-L1 proteins are detected in most of the cell lines, but not in A549 and THP-1 cells by the two independent antibodies. 

siRNA Knockdown Validation (Figure 3): Anti-PD-L1 antibody (4059) specificity was further verified by PD-L1 specific siRNA knockdown. PD-L1 signal in HeLa cells transfected with PD-L1 siRNAs was much weaker in comparison with that in HeLa cells transfected with control siRNAs.

Overexpression Validation (Figure 4): Anti-PD-L1 antibodies (4059) can detect the overexpression of PD-L1 protein in 293 cells transfected with PD-L1.

Isoforms:
Human PD-L1 has 3 isoforms, including isoform 1 (290aa, 33.3kD), isoform 2 (176aa, 20.2kD), and isoform 3 (178aa, 20.5kD). This antibody detects human isoform 1&3, mouse and rat PD-L1 (290aa, 33kD for both of them).
Purification:
PD-L1 Antibody is affinity chromatography purified via peptide column.
Clonality:
Polyclonal
Isotype:
IgG
physical-state:
Liquid
Buffer:
PD-L1 Antibody is supplied in PBS containing 0.02% sodium azide.
Concentration:
1 mg/mL
Storage Conditions:
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.
Ncbi Official Symbol:
CD274
Additional Names:
PD-L1 Antibody: B7-H, B7H1, PDL1, PD-L1, PDCD1L1, PDCD1LG1, Programmed cell death 1 ligand 1, B7 homolog 1
Protein Accession Number:
NP_054862
Protein Gi Number:
7661534
Ncbi Gene Id Number:
29126
User Note:
Optimal dilutions for each application to be determined by the researcher.
Background:
PD-L1 plays a critical role in induction and maintenance of immune tolerance to self. As a ligand for the inhibitory receptor PDCD1/CD279, PD-L1 modulates the activation threshold of T-cells and limits T-cell effector response (1). The PDCD1/CD279-mediated inhibitory pathway is exploited by tumors to attenuate anti-tumor immunity and facilitate tumor survival (2,3). Through a yet unknown activating receptor, it may costimulate T-cell subsets that predominantly produce interleukin-10 (IL10) (4).
Background Reference 1:
Freeman et al. Exp. Med. 2000; 192:1027-34.
Background Reference 2:
Burr et al. Nature 2017; 549:101-5.
Background Reference 3:
Mezzadra et al. Nature 2017; 549:106-10.
Background Reference 4:
Dong et al. Nat. Med. 1999 5:1365-9.
Citation 1:
Dhar et al. Evaluation of PD-L1 expression on vortex-isolated circulating tumor cells in metastatic lung cancer. Sci Rep. 2018;8(1):2592. PMID: 29416054
Citation 2:
Gadiot et al. Overall survival and PD-L1 expression in metastasized malignant melanoma. Cancer.2011;117(10):2192-201. PMID: 21523733
Citation 3:
Angell et al. BRAF V600E in papillary thyroid carcinoma is associated with increased programmed death ligand 1 expression and suppressive immune cell infiltration. Thyroid. 2014; 24(9):1385-93PMID: 24955518
Citation 4:
Ilie et al. Comparative study of the PD-L1 status between surgically resected specimens and matched biopsies of NSCLC patients reveal major discordances: a potential issue for anti-PD-L1 therapeutic strategies. Ann Oncol. 2016;27(1):147-53.PMID: 26483045
Citation 5:
Heymann et al. PD-L1 expression in non-small cell lung carcinoma: Comparison among cytology, small biopsy, and surgical resection specimens. Cancer Cytopathol. 2017;125(12):896-907. PMID: 29024471
Citation 6:
Hamanishi et al. Safety and Antitumor Activity of Anti-PD-1 Antibody, Nivolumab, in Patients With Platinum-Resistant Ovarian Cancer. J Clin Oncol. 2015; 33(34):4015-22. PMID: 26351349
Citation 7:
Taube et al. Colocalization of inflammatory response with B7-h1 expression in human melanocytic lesions supports an adaptive resistance mechanism of immune escape. Sci Transl Med. 2012;4(127):127ra37. PMID: 22461641
Citation 8:
Munari et al. PD-L1 Expression Heterogeneity in Non-Small Cell Lung Cancer: Defining Criteria for Harmonization between Biopsy Specimens and Whole Sections. J Thorac Oncol. 2018;13(8): 1113-1120. PMID: 29704674
Citation 9:
Li et al. Comparison of 22C3 PD-L1 Expression between Surgically Resected Specimens and Paired Tissue Microarrays in Non-Small Cell Lung Cancer. J Thorac Oncol. 2017;12(10):1536-1543. PMID: 28751245
Citation 10:
Shen et al. Programmed cell death ligand 1 expression in osteosarcoma. Cancer Immunol Res. 2014;2(7):690-698. PMID: 24866169
Citation 11:
Mimura et al. PD-L1 expression is mainly regulated by interferon gamma associated with JAK-STAT pathway in gastric cancer. Cancer Sci. 2018 ;109(1):43-53.PMID: 29034543
Citation 12:
Habib et al. PDL-1 Blockade Prevents T Cell Exhaustion, Inhibits Autophagy, and Promotes Clearance of Leishmania donovani. Infect Immun. 2018;86(6). PMID: 29610255
Citation 13:
Gonin et al. Expression of IL-27 by tumor cells in invasive cutaneous and metastatic melanomas. PLoS One. 2013;8(10):e75694. PMID: 24130734
Citation 14:
Gagné et al. Comprehensive Assessment of PD-L1 Staining Heterogeneity in Pulmonary Adenocarcinomas Using Tissue Microarrays: Impact of the Architecture Pattern and the Number of Cores. Am J Surg Pathol. 2018;42(5):687-694.PMID: 29309297
Citation 15:
Teixidó et al. Assays for predicting and monitoring responses to lung cancer immunotherapy. Cancer Biol Med. 2015;12(2):87-95. PMID: 26175924
Citation 16:
Ohue et al. Survival of Lung Adenocarcinoma Patients Predicted from Expression of PD-L1, Galectin-9, and XAGE1 (GAGED2a) on Tumor Cells and Tumor-Infiltrating T Cells. Cancer Immunol Res. 2016;4(12):1049-1060. PMID: 27799141
Citation 17:
Kao et al. Assessing the Effects of Concurrent versus Sequential Cisplatin/Radiotherapy on Immune Status in Lung Tumor-Bearing C57BL/6 Mice. Cancer Immunol Res. 2015;3(7):741-50. PMID: 25672395
Citation 18:
Fujimura et al. Potential use of bisphosphonates in invasive extramammary Paget's disease: an immunohistochemical Investigation. Clin Dev Immunol. 2013 ;2013:164982. PMID: 23606867
Citation 19:
Sakata et al. Comparison of Programmed Death Ligand-1 Immunohistochemical Staining Between Endobronchial Ultrasound Transbronchial Needle Aspiration and Resected Lung Cancer Specimens. Chest. 2018;154(4):827-837. PMID: 30059678
Citation 20:
Kitazono et al. Reliability of Small Biopsy Samples Compared With Resected Specimens for the Determination of Programmed Death-Ligand 1 Expression in Non--Small-Cell Lung Cancer. Clin Lung Cancer. 2015 ;16(5):385-90.PMID: 25937270

FOR RESEARCH USE ONLY

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Disclaimer:
Optimal dilutions/concentrations should be determined by the end user. The information provided is a guideline for product use. This product is for research use only.

CATALOG NUMBER:

4059

List Size:
0.02 mg, 0.1 mg

List Price:

Price range: $99.00 through $445.00

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