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<strong>Figure 1 Overexpression Validation of PD-L1 in 293 Cells </strong><br>Loading: 15 μg of lysates per lane.Antibodies: RF16035 (A, 0.25 μg/mL; B, 0.5 μg/mL), 1 h incubation at RT in 5% NFDM/TBST.Secondary: Goat anti-mouse IgG HRP conjugate at 1:5000 dilution.
<strong>Figure 2 Independent Antibody Validation (IAV) via Protein Expression Profile in 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: RF16035 (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 Western Blot Validation of PD-L1 in Raji Cells </strong><br> Loading: Lysates/proteins at 15 μg per lane.Antibodies: RF16035 (4 μg/mL). 1 h incubation at RT in 5% NFDM/TBST.Secondary: Goat anti-mouse IgG HRP conjugate at 1:10000 dilution.
<strong>Figure 5 Immunofluorescence Validation of PD-L1 in Transfected 293 Cells</strong><br> Immunofluorescent analysis of 4% paraformaldehyde-fixed PD-L1 transfected 293 cells labeling PD-L1 with RF16035 at 2 μg/mL, followed by goat anti-mouse IgG secondary antibody at 1/500 dilution (red) and DAPI staining (blue).
<strong>Figure 6 Immunofluorescence Validation of PD-L1 in Human Stomach Carcinoma Tissue</strong><br> Immunofluorescent analysis of 4% paraformaldehyde-fixed human stomach carcinoma tissue labeling PD-L1 with RF16035 at 2 μg/mL, followed by goat anti-mouse IgG secondary antibody at 1/500 dilution (red) and DAPI staining (blue).
<strong>Figure 7 Immunofluorescence Validation of PD-L1 in Human Tonsil Tissue</strong><br> Immunofluorescent analysis of 4% paraformaldehyde-fixed human tonsil tissue labeling PD-L1 with RF16035 at 2 μg/mL, followed by goat anti-mouse IgG secondary antibody at 1/500 dilution (red) and DAPI staining (blue).
<strong>Figure 8 Immunohistochemistry Validation of PD-L1 in Human Stomach Carcinoma Tissue </strong><br>Immunohistochemical analysis of paraffin-embedded human stomach carcinoma tissue using anti-PD-L1 antibody (RF16035) 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-mouse IgG H&L (HRP) at 1/250 was used as secondary. Counter stained with Hematoxylin. Lower left: Use mouse IgG antibody at 1 μg/ml as control.
<strong>Figure 9 Immunohistochemistry Validation of PD-L1 in Human Tonsil Carcinoma Tissue </strong><br>Immunohistochemical analysis of paraffin-embedded human tonsil tissue using anti-PD-L1 antibody (RF16035) 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-mouse IgG H&L (HRP) at 1/250 was used as secondary. Counter stained with Hematoxylin. Lower left: Use mouse IgG antibody at 1 μg/ml as control.
<strong> Figure 10 Immunocytochemistry Validation of PD-L1 in PD-L1 Overexpressed 293 Cells </strong><br>Immunocytochemical analysis of PD-L1 transfected 293 cells using anti- PD-L1 antibody (RF16035) at 1 μg/mL. Cells 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. Lower left: Use mouse IgG antibody at 1 μg/mL as control.
Figure 1 Overexpression Validation of PD-L1 in 293 Cells
Loading: 15 μg of lysates per lane.Antibodies: RF16035 (A, 0.25 μg/mL; B, 0.5 μg/mL), 1 h incubation at RT in 5% NFDM/TBST.Secondary: Goat anti-mouse IgG HRP conjugate at 1:5000 dilution.
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PDL1 Antibody [6H10]

CATALOG NUMBER: RF16035

Clonality:
Monoclonal
Tested Applications:
ELISA, ICC, IF, IHC-P, WB
Host Species:
Mouse
Species Reactivity:
Human, Mouse
Conjugate:
Unconjugated
Specifications
Host Species:
Mouse
Species Reactivity:
Human, Mouse
Homology:
Predicted species reactivity based on immunogen sequence: Rat: (77%), Mouse: (71%)
Immunogen:
Anti-PD-L1 antibody (RF16035) was raised against the extracellular domain of human PD-L1.
Conjugate:
Unconjugated
Tested Applications:
ELISA, ICC, IF, IHC-P, WB
Application Note:
WB: 0.25 - 4 μg/mL; IF: 2 μg/mL; IHC-P: 5 μg/mL; ICC: 1 μg/mL.
Antibody validated: Western Blot in human and mouse samples; Immunohistochemistry, Immunocytochemistry and Immunofluorescence in human 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. 1340 - Human Stomach Carcinoma Tissue Lysate
Positive Control 4:
Cat. No. 1315 - Human Tonsil Tissue
Predicted Molecular Weight:
Predicted: 33kD
Observed: 37kD
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, RF16035 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 (RF16035) 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 1, 5, 10): Anti-PD-L1 antibodies (RF16035) 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 supplied as protein A purified IgG1.
Clonality:
Monoclonal
Isotype:
IgG1
physical-state:
Liquid
Buffer:
PD-L1 Antibody is supplied in PBS containing 0.02% sodium azide and 50% glycerol.
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: Programmed cell death 1 ligand-1, programmed death ligand 1, PDL1, PDL-1, B7-H1
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.

FOR RESEARCH USE ONLY

For additional information, visit ProSci’s Terms & Conditions Page.

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:

RF16035

List Size:
0.02 mg, 0.1 mg

List Price:

Price range: $99.00 through $445.00

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