Name: Anti-PD-L1 antibody [EPR19759]
SKU: ab213524
Rating: 5 (1 reviews)

Anti-PD-L1 antibody [EPR19759] is a rabbit recombinant monoclonal antibody that is used to detect PD-L1 in Flow cytometry (Intra), ICC/IF, IHC-P, IP, Western blot. Suitable for Human samples.

- Specificity confirmed with CD274 knockout cell line validation
- Using biophysical QC, antibody identity is confirmed at a molecular level for unrivaled batch-batch consistency
- Cited in over 90 publications

Related conjugates and formulations

ab215251
Conjugated
Alexa Fluor® 647 Anti-PD-L1 antibody [EPR19759]
ab216742
Conjugated
Alexa Fluor® 594 Anti-PD-L1 antibody [EPR19759]
ab220314
Conjugated
Alexa Fluor® 568 Anti-PD-L1 antibody [EPR19759]
ab221612
Carrier free
Anti-PD-L1 antibody [EPR19759] - BSA and Azide free
ab215349
Conjugated
HRP Anti-PD-L1 antibody [EPR19759]
ab214958
Conjugated
Alexa Fluor® 488 Anti-PD-L1 antibody [EPR19759]

Images

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PD-L1 antibody [EPR19759] (AB213524), expandable thumbnail

Publications

Key facts

Isotype: IgG
Host species: Rabbit
Storage buffer: pH: 7.2 - 7.4
Preservative: 0.01% Sodium azide
Constituents: 59% PBS, 40% Glycerol (glycerin, glycerine), 0.05% BSA
Form: Liquid
Clonality: Monoclonal

Immunogen

The exact immunogen used to generate this antibody is proprietary information.

Reactivity data

Select an application

Product promiseTestedExpectedPredictedNot recommended

	IHC-P	IP	WB	ICC/IF	Flow Cyt (Intra)	Flow Cyt
Human	Tested	Tested	Tested	Tested	Expected	Not recommended
Mouse	Not recommended	Not recommended	Not recommended	Not recommended	Not recommended	Not recommended
Recombinant full length protein - Human	Not recommended	Not recommended	Tested	Not recommended	Not recommended	Not recommended
Transfected cell line - Chinese hamster	Not recommended	Not recommended	Not recommended	Tested	Tested	Not recommended

Tested
Tested

Species	Dilution info	Notes
Species Human	Dilution info 1/250	Notes Antigen retrieval: Universal HIER antigen retrieval reagent (ab208572). Perform heat-mediated antigen retrieval before commencing with IHC staining protocol.

Not recommended
Not recommended

Species	Dilution info	Notes
Species Recombinant full length protein - Human, Transfected cell line - Chinese hamster, Mouse	Dilution info -	Notes -

Tested
Tested

Species	Dilution info	Notes
Species Human	Dilution info 1/30	Notes -

Not recommended
Not recommended

Species	Dilution info	Notes
Species Recombinant full length protein - Human, Transfected cell line - Chinese hamster, Mouse	Dilution info -	Notes -

Tested
Tested

Species	Dilution info	Notes
Species Recombinant full length protein - Human	Dilution info 1/1000	Notes Anti-PD-L1 antibody [73-10] ab228415 works better than this product in western blot testing.
Species Human	Dilution info 1/1000	Notes Anti-PD-L1 antibody [73-10] ab228415 works better than this product in western blot testing.

Not recommended
Not recommended

Species	Dilution info	Notes
Species Transfected cell line - Chinese hamster, Mouse	Dilution info -	Notes -

Tested
Tested

Species	Dilution info	Notes
Species Transfected cell line - Chinese hamster	Dilution info 1/500	Notes -
Species Human	Dilution info 1/500	Notes -

Not recommended
Not recommended

Species	Dilution info	Notes
Species Recombinant full length protein - Human, Mouse	Dilution info -	Notes -

Tested
Tested

Species	Dilution info	Notes
Species Transfected cell line - Chinese hamster	Dilution info 1/500	Notes -

Expected
Expected

Species	Dilution info	Notes
Species Human	Dilution info Use at an assay dependent concentration.	Notes -

Not recommended
Not recommended

Species	Dilution info	Notes
Species Recombinant full length protein - Human, Mouse	Dilution info -	Notes -

Not recommended
Not recommended

Species	Dilution info	Notes
Species Mouse, Human, Recombinant full length protein - Human, Transfected cell line - Chinese hamster	Dilution info -	Notes -

Associated Products

Select an associated product type

15 products for Alternative Product

1 product for Alternative Version

Target data

See full target information CD274

Function

Plays a critical role in induction and maintenance of immune tolerance to self (PubMed:11015443, PubMed:28813410, PubMed:28813417, PubMed:31399419). As a ligand for the inhibitory receptor PDCD1/PD-1, modulates the activation threshold of T-cells and limits T-cell effector response (PubMed:11015443, PubMed:28813410, PubMed:28813417, PubMed:36727298). Through a yet unknown activating receptor, may costimulate T-cell subsets that predominantly produce interleukin-10 (IL10) (PubMed:10581077). Can also act as a transcription coactivator: in response to hypoxia, translocates into the nucleus via its interaction with phosphorylated STAT3 and promotes transcription of GSDMC, leading to pyroptosis (PubMed:32929201). The PDCD1-mediated inhibitory pathway is exploited by tumors to attenuate anti-tumor immunity and escape destruction by the immune system, thereby facilitating tumor survival (PubMed:28813410, PubMed:28813417). The interaction with PDCD1/PD-1 inhibits cytotoxic T lymphocytes (CTLs) effector function (By similarity). The blockage of the PDCD1-mediated pathway results in the reversal of the exhausted T-cell phenotype and the normalization of the anti-tumor response, providing a rationale for cancer immunotherapy (By similarity).

Alternative names

CD274, B7H1, PDCD1L1, PDCD1LG1, PDL1, Programmed cell death 1 ligand 1, PD-L1, PDCD1 ligand 1, Programmed death ligand 1, hPD-L1, B7 homolog 1, B7-H1

Key facts

Isotype: IgG
Host species: Rabbit
Storage buffer: pH: 7.2 - 7.4
Preservative: 0.01% Sodium azide
Constituents: 59% PBS, 40% Glycerol (glycerin, glycerine), 0.05% BSA
Form: Liquid
Clonality: Monoclonal
Immunogen: The exact immunogen used to generate this antibody is proprietary information.
Clone number: EPR19759
Purification technique: Affinity purification Protein A
Specificity: FURTHER INFORMATION ON SPECIFICITY (Chinese Version) available under the support & downloads section.
Concentration

Storage

Shipped at conditions: Blue Ice
Appropriate short-term storage duration: 1-2 weeks
Appropriate short-term storage conditions: +4°C
Appropriate long-term storage conditions: -20°C
Aliquoting information: Upon delivery aliquot
Storage information: Avoid freeze / thaw cycle

Notes

Anti-PD-L1 antibody [EPR19759] (ab213524) was developed by Abcam using patented rabbit monoclonal antibody technology and is validated for use in Flow Cyt (Intra), ICC/IF, IHC-P, IP, WB in human samples.

Anti-PD-L1 antibody [EPR19759] (ab213524) has been cited over 96 times in peer reviewed journals and is trusted by the scientific community.

Abcams high quality manufacturing and validation processes ensure Anti-PD-L1 antibody [EPR19759] (ab213524) has high sensitivity and specificity alongside high lot-to-lot consistency and reproducibility.

The specificity of Anti-PD-L1 antibody [EPR19759] (ab213524) has been confirmed by testing in knockout samples.

Anti-PD-L1 antibody [EPR19759] (ab213524) specifically detects PD-L1 (UniProt ID: Q9NZQ7; Molecular weight: 31kDa) and is sold in 100 uL and 1 mL selling sizes.

Conjugation-ready, carrier free format available for antibody clone EPR19759 - Anti-PD-L1 antibody [EPR19759] - BSA and Azide free ab221612.

Antibody clone EPR19759 is also available pre-conjugated to a variety of labels for your convenience - Alexa Fluor® 488, Alexa Fluor® 647, HRP, Alexa Fluor® 594, Alexa Fluor® 568 (Alexa Fluor® 488 Anti-PD-L1 antibody [EPR19759] ab214958, Alexa Fluor® 647 Anti-PD-L1 antibody [EPR19759] ab215251, HRP Anti-PD-L1 antibody [EPR19759] ab215349, Alexa Fluor® 594 Anti-PD-L1 antibody [EPR19759] ab216742, ab22314).

Programmed death-ligand 1 (PD-L1) is a critical protein in immuno-oncology, playing a significant role in tumor immune evasion. It is expressed on the surface of tumor cells and binds to the PD-1 receptor on T cells, inhibiting their activity and allowing tumors to escape immune detection.

Our RabMAb® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMAb® patents.

This product is a recombinant monoclonal antibody, which offers several advantages including:

- High batch-to-batch consistency and reproducibility
- Improved sensitivity and specificity
- Long-term security of supply
- Animal-free batch production

For more information, read more on recombinant antibodies.

Supplementary info

This supplementary information is collated from multiple sources and compiled automatically.

Activity summary

PD-L1 also known as Programmed Death-Ligand 1 or CD274 is a protein involved in immune modulation. Mechanically PD-L1 interacts with its receptors particularly PD-1 to regulate cellular immune responses. This transmembrane protein has a calculated molecular weight of approximately 33 kDa. PD-L1 is expressed on various cell types including tumor cells and immune cells such as dendritic cells macrophages and B cells. Its expression is often upregulated in response to inflammatory cytokines.

Biological function summary

PD-L1 plays a central role in immune evasion mechanisms utilized by tumors. It is not part of a larger protein complex but directly interacts with PD-1 and CD80. When PD-L1 binds to PD-1 it sends inhibitory signals leading to decreased T cell activation and proliferation allowing cancer cells to avoid immune destruction. PD-L1 expression provides a mechanism for tumors to suppress immune surveillance facilitating tumor progression.

Pathways

PD-L1 is integral to the immune checkpoint pathway which is an important regulator of immune response. The interaction between PD-L1 and PD-1 provides a mechanism for immune tolerance which is part of the broader adaptive immune system pathway. PD-L1 is related to other immune checkpoint proteins such as CTLA-4 in its function to limit autoreactivity and promote immune homeostasis.

Associated diseases and disorders

PD-L1 is most associated with cancer particularly in tumors such as melanoma and non-small cell lung cancer. PD-L1 expression on tumor cells often correlates with poor prognosis. PD-L1 directly interacts with PD-1 in these cancers a target for immunotherapies such as checkpoint inhibitors which aim to block this interaction and restore immune activity against tumors. PD-L1 involvement extends to autoimmune disorders where altered expression can impact tolerance and lead to immune-related tissue damage.

Product promise

We are dedicated to supporting your work with high quality reagents and we are here for you every step of the way should you need us.

In the unlikely event of one of our products not working as expected, you are covered by our product promise.

Full details and terms and conditions can be found here:
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18 product images

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PD-L1 antibody [EPR19759] (ab213524)
Immunohistochemical analysis of paraffin-embedded human stomach cancer tissue labeling PD-L1 with ab213524 at 1/250 dilution, followed by Rabbit specific IHC polymer detection kit HRP/DAB (Rabbit specific IHC polymer detection kit HRP/DAB ab209101).
Membrane staining on the human stomach cancer is observed.
Counter stained with Hematoxylin.
Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is rabbit specific IHC polymer detection kit HRP/DAB (Rabbit specific IHC polymer detection kit HRP/DAB ab209101).
Perform heat mediated antigen retrieval before commencing with IHC staining protocol.
Western blot - Anti-PD-L1 antibody [EPR19759] (ab213524)
Lanes 1- 6: Merged signal (red and green). Green - ab213524 observed at 50 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (Anti-GAPDH antibody [6C5] - Loading Control ab8245) observed at 37 kDa.
ab213524 was shown to react with PD-L1 in wild-type A549 treated with 100 ng/ml IFN gamma for 48 h cells in western blot. Loss of signal was observed when both treated and untreated knockout cell lines Human CD274 (PD-L1) knockout A549 cell line ab267054 ( treated and untreated knockout cell lysates Human CD274 (PD-L1) knockout A549 cell lysate ab256831) were used. Wild-type A549 treated with 100 ng/ml IFN gamma for 48 h and CD274 knockout A549 treated with 100 ng/ml IFN gamma for 48 h cell lysates were subjected to SDS-PAGE. Membrane was blocked for 1 hour at room temperature in 0.1% TBST with 3% non-fat dried milk. ab213524 and Anti-GAPDH antibody [6C5] - Loading Control (Anti-GAPDH antibody [6C5] - Loading Control ab8245) were incubated overnight at 4°C at a 1 in 1000 dilution and a 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^®800CW) preadsorbed (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) and Goat anti-Mouse IgG H&L (IRDye^®680RD) preadsorbed (Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.
Lane 1: Wild-type A549 treated with 100 ng/ml IFN gamma (Recombinant Human Interferon gamma protein (Active) ab259377) for 48 h cell lysate at 20 µg
Lanes 2 and 6: CD274 knockout A549 treated with 100 ng/ml IFN gamma (Recombinant Human Interferon gamma protein (Active) ab259377) for 48 h cell lysate at 20 µg
Lane 2: Western blot - Human CD274 (PD-L1) knockout A549 cell line (Human CD274 (PD-L1) knockout A549 cell line ab267054)
Lane 3: U-87 MG cell lysate at 20 µg
Lane 4: MCF7 cell lysate at 20 µg
Lane 5: Wild-type A549 untreated cell lysate at 20 µg
Performed under reducing conditions.
Predicted band size: 33 kDa
Observed band size: 50 kDa
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PD-L1 antibody [EPR19759] (ab213524)
Anti-PD-L1 antibody [EPR19759] (ab213524)
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human tonsil tissue labelling PD-L1 with ab213524 at a dilution of 1:250. Heat mediated antigen retrieval was performed using AR9 antigen retrieval solution, and microwave treatment for 15 min at 20% power. Anti-Rabbit/Mouse HRP polymer (PerkinElmer Opal Polymer HRP Ms Plus Rb) was used as secondary antibody. Opal tyramide amplification was performed using Opal 520 fluorophore. Counterstained with DAPI stain. Image scanned with Vectra 3.0 and analyzed via Phenochart software.
This image was courteously provided by Dr. Houssein Abdul Sater, Georgia Cancer Center.
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PD-L1 antibody [EPR19759] (ab213524)
Immunohistochemical analysis of paraffin-embedded human tonsil tissue labeling PD-L1 with ab213524 at 1/250 dilution, followed by Rabbit specific IHC polymer detection kit HRP/DAB (Rabbit specific IHC polymer detection kit HRP/DAB ab209101).
Membrane staining on the human tonsil crypt epithelium is observed.
Counter stained with Hematoxylin.
Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is rabbit specific IHC polymer detection kit HRP/DAB (Rabbit specific IHC polymer detection kit HRP/DAB ab209101).
Perform heat mediated antigen retrieval before commencing with IHC staining protocol.
Immunocytochemistry/ Immunofluorescence - Anti-PD-L1 antibody [EPR19759] (ab213524)
Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized CHO (Chinese hamster ovary cell line) cells labeling PD-L1 with ab213524 at 1/100 dilution, followed by Goat Anti-Rabbit IgG (Alexa Fluor^® 488) (Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077) secondary antibody at 1/1000 dilution (green).
Confocal image showing membrane and cytoplasmic staining on CHO-PDL1 cells.
The nuclear counterstain is DAPI (blue).
Tubulin is detected with Anti-alpha Tubulin mouse MAb (Anti-alpha Tubulin antibody [DM1A] - Loading Control ab7291) at 1/1000 dilution, followed by Goat Anti-Mouse IgG H&L (Alexa Fluor^® 594) (Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) preadsorbed ab150120) secondary antibody at 1/1000 dilution (red).
The negative controls are as follows:-
-ve control 1: ab213524 at 1/100 dilution, followed by Goat Anti-Mouse IgG H&L (Alexa Fluor^® 594) (Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) preadsorbed ab150120) secondary antibody at 1/1000 dilution.
-ve control 2: Anti-alpha Tubulin mouse MAb (Anti-alpha Tubulin antibody [DM1A] - Loading Control ab7291) at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077) secondary antibody at 1/1000 dilution.
Western blot - Anti-PD-L1 antibody [EPR19759] (ab213524)
Lanes 1 - 6: Merged signal (red and green). Green - ab213524 observed at 50 kDa. Red - loading control, Anti-GAPDH antibody [6C5] - Loading Control ab8245 observed at 37 kDa.
ab213524 Recombinant Anti-PD-L1 antibody [EPR19759] was shown to specifically react with PD-L1 in wild-type A549 treated with 100 ng/mL IFN gamma for 48 h cells in western blot. Loss of signal was observed when both treated and untreated knockout cell line Human CD274 (PD-L1) knockout A549 cell line ab267055 (treated and untreated knockout cell lysates Human CD274 (PD-L1) knockout A549 cell lysate ab256866) were used. Wild-type and PD-L1 knockout samples were subjected to SDS-PAGE. Membrane was blocked for 1 hour at room temperature in 0.1% TBST with 3% non-fat dried milk. ab213524 and Anti-GAPDH antibody [6C5] - Loading Control (Anti-GAPDH antibody [6C5] - Loading Control ab8245) were incubated overnight at 4°C at 1 in 1000 and 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preadsorbed (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preadsorbed (Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.
All lanes: Western blot - Anti-PD-L1 antibody [EPR19759] (ab213524) at 1/1000 dilution
Lane 1: Wild-type A549 treated with 100 ng/mL IFN gamma for 48 h cell lysate at 20 µg
Lane 2: CD274 knockout A549 treated with 100 ng/mL IFN gamma for 48 h cell lysate at 20 µg
Lane 2: Western blot - Human CD274 (PD-L1) knockout A549 cell line (Human CD274 (PD-L1) knockout A549 cell line ab267055)
Lane 3: U-87 MG cell lysate at 20 µg
Lane 4: MCF7 cell lysate at 20 µg
Lane 5: Wild-type A549 untreated cell lysate at 20 µg
Lane 6: CD274 knockout A549 untreated cell lysate at 20 µg
Performed under reducing conditions.
Predicted band size: 33 kDa
Observed band size: 50 kDa
Western blot - Anti-PD-L1 antibody [EPR19759] (ab213524)
Expression of PD-L1 varied widely among the tumor cell lines
Blocking buffer and concentration : 5% NFDM/TBST
Diluting buffer and concentration : 5% NFDM/TBST
All lanes: Western blot - Anti-PD-L1 antibody [EPR19759] (ab213524) at 1/1000 dilution
Lane 1: H1975 (Human non-small cell lung cancer epithelial cell) whole cell lysate at 20 µg
Lane 2: NCI-H1299 (Human lung carcinoma epithelial cell) whole cell lysate at 20 µg
Lane 3: A549 (Human lung carcinoma epithelial cell) whole cell lysate at 20 µg
Lane 4: MDA-MB-231 (Human breast adenocarcinoma epithelial cell) whole cell lysate at 20 µg
Lane 5: MCF7 (Human breast adenocarcinoma epithelial cell) whole cell lysate at 20 µg
Lane 6: SK-BR-3 (Human breast adenocarcinoma epithelial cell) whole cell lysate at 20 µg
Lane 7: SW480 (Human colorectal adenocarcinoma epithelial cell) whole cell lysate at 20 µg
Lane 8: HCT 116 (Human colorectal carcinoma epithelial cell) whole cell lysate at 20 µg
Lane 9: PC-3 (Human prostate adenocarcinoma epithelial cell) whole cell lysate at 20 µg
Lane 10: DU 145 (Human prostate carcinoma epithelial cell) whole cell lysate at 20 µg
Lane 11: A375 (Human malignant melanoma epithelial cell) whole cell lysate at 20 µg
Lane 12: MeWo (Human malignant melanoma fibroblast) whole cell lysate at 20 µg
Lane 13: U-87 MG (Human glioblastoma-astrocytoma epithelial cell) whole cell lysate at 20 µg
Lane 14: Huh7 (Human hepatocellular carcinoma epithelial cell) whole cell lysate at 20 µg
Lane 15: HepG2(Human hepatocellular carcinoma epithelial cell) whole cell lysate at 20 µg
Lane 16: BXPC-3 (Human pancreas adenocarcinoma epithelial cell) whole cell lysate at 20 µg
Lane 17: PANC-1 (Human pancreatic epithelioid carcinoma epithelial cell) whole cell lysate at 20 µg
Lane 18: NIH:OVCAR-3 (Human ovary adenocarcinoma epithelial cell) whole cell lysate at 20 µg
Lane 19: SK-OV-3 (Human ovarian cancer epithelial cell) whole cell lysate at 20 µg
Lane 20: HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate at 20 µg
Secondary
All lanes: Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated at 1/20000 dilution
Developed using the ECL technique.
Predicted band size: 33 kDa
Observed band size: 40-60 kDa
Exposure time: 180s
Western blot - Anti-PD-L1 antibody [EPR19759] (ab213524)
Blocking and diluting buffer: 5% NFDM/TBST.
All lanes: Western blot - Anti-PD-L1 antibody [EPR19759] (ab213524) at 0.5 µg/mL
Lane 1: Untreated A549 (Human lung carcinoma epithelial cell) whole cell lysate
Lane 2: A549 (Human lung carcinoma epithelial cell) treated with 100ng/ml Interferon gamma for 48 hours whole cell lysate
Secondary
All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/20000 dilution
Predicted band size: 33 kDa
Exposure time: 3s
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PD-L1 antibody [EPR19759] (ab213524)
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of Human spleen tissue labelling PDI with Anti-PD1 antibody [EPR23119-111] ab243644 at 1.02 μg/mL (B), PD-L 1 with ab213524 at 1/100 dilution (C) and CD68 with Anti-CD68 antibody [EPR20545] ab213363 at 1/300 dilution (D). Anti-Rabbit and Mouse Polymer HRP was used as a secondary antibody, and DAPI was used for a nuclear counter stain. Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20mins. Heat mediated antigen retrieval (Leica ER2, PH9.0, 20 minutes) was used in between rounds of tyramide signal amplification to remove the antibodies from the previous round, to avoid any cross-reactivity.
Panel A: merged staining of anti- PD1 (green, Opal™520), anti- PD-L1 (red, Opal™570) and anti- CD68 (yellow, Opal™690).
Panel B: Anti- PD1 stained on antigen-stimulated T cells.
Panel C: anti- PD-L1 stained on cells involved in T cell inhibition
Panel D: anti-CD68 stained on macrophages.
The section was incubated in three rounds of staining: in the order of Anti-PD1 antibody [EPR23119-111] ab243644, Anti-CD68 antibody [EPR20545] ab213363 and ab213524 for 30 mins at room temperature. Each round was followed by a separate fluorescent tyramide signal amplification system.
The immunostaining was performed on a Leica Biosystems BOND® RX instrument with an Opal™ 4-color kit. Image acquisition was performed with Leica SP8 confocal microscope.
Flow Cytometry (Intracellular) - Anti-PD-L1 antibody [EPR19759] (ab213524)
Intracellular Flow Cytometry analysis of CHO-PD-L1 (red) and CHO-S (blue) cells labelling PD-L1 with ab213524 at 1/500. Cells were fixed with 4% paraformaldehyde, permeabilized with 0.1% tween-20-PBS and blocked with 10% goat serum. An Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/2000) was used as the secondary antibody.
Immunoprecipitation - Anti-PD-L1 antibody [EPR19759] (ab213524)
PD-L1 was immunoprecipitated from 0.35 mg of NCI-H1975 (Human non-small cell lung cancer cell line) whole cell lysate with ab213524 at 1/30 dilution.
Western blot was performed from the immunoprecipitate using ab213524 at 1/1000 dilution.
VeriBlot for IP Detection Reagent (HRP) (VeriBlot for IP Detection Reagent (HRP) ab131366), was used for detection at 1/10,000 dilution.
Lane 1: NCI-H1975 whole cell lysate 10μg (Input).
Lane 2: ab213524 IP in NCI-H1975 whole cell lysate.
Lane 3: Rabbit monoclonal IgG (Rabbit IgG, monoclonal [EPR25A] - Isotype Control ab172730) instead of ab213524 in NCI-H1975 whole cell lysate.
Blocking and dilution buffer and concentration: 5% NFDM/TBST.
Exposure time: 3 minutes.
All lanes: Immunoprecipitation - Anti-PD-L1 antibody [EPR19759] (ab213524)
Predicted band size: 33 kDa
Immunocytochemistry/ Immunofluorescence - Anti-PD-L1 antibody [EPR19759] (ab213524)
Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized NCI-H1975 (Human lung non small cell carcinoma cell line) cells labeling PD-L1 with ab213524 at 1/100 dilution, followed by Goat Anti-Rabbit IgG (Alexa Fluor^® 488) (Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077) secondary antibody at 1/1000 dilution (green).
Confocal image showing weakly membrane and cytoplasmic staining on NCI-H1975 cells.
The nuclear counterstain is DAPI (blue).
Tubulin is detected with Anti-alpha Tubulin mouse MAb (Anti-alpha Tubulin antibody [DM1A] - Loading Control ab7291) at 1/1000 dilution, followed by Goat Anti-Mouse IgG H&L (Alexa Fluor^® 594) (Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) preadsorbed ab150120) secondary antibody at 1/1000 dilution (red).
The negative controls are as follows:-
-ve control 1: ab213524 at 1/100 dilution followed by Goat Anti-Mouse IgG H&L (Alexa Fluor^® 594) (Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) preadsorbed ab150120) secondary antibody at 1/1000 dilution.
-ve control 2: Anti-alpha Tubulin mouse MAb (Anti-alpha Tubulin antibody [DM1A] - Loading Control ab7291) at 1/1000 dilution followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077) secondary antibody at 1/1000 dilution.
Western blot - Anti-PD-L1 antibody [EPR19759] (ab213524)
Lanes 1- 6: Merged signal (red and green). Green - ab213524 observed at 50 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (Anti-GAPDH antibody [6C5] - Loading Control ab8245) observed at 37 kDa.
ab213524 was shown to react with PD-L1 in wild-type A549 treated with 100 ng/ml IFN gamma for 48 h cells in western blot. Loss of signal was observed when both treated and untreated knockout cell lines Human CD274 (PD-L1) knockout A549 cell line ab267054 ( treated and untreated knockout cell lysates Human CD274 (PD-L1) knockout A549 cell lysate ab256831) were used. Wild-type A549 treated with 100 ng/ml IFN gamma for 48 h and CD274 knockout A549 treated with 100 ng/ml IFN gamma for 48 h cell lysates were subjected to SDS-PAGE. Membrane was blocked for 1 hour at room temperature in 0.1% TBST with 3% non-fat dried milk. ab213524 and Anti-GAPDH antibody [6C5] - Loading Control (Anti-GAPDH antibody [6C5] - Loading Control ab8245) were incubated overnight at 4°C at a 1 in 1000 dilution and a 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^®800CW) preadsorbed (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) and Goat anti-Mouse IgG H&L (IRDye^®680RD) preadsorbed (Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.
All lanes: Western blot - Anti-PD-L1 antibody [EPR19759] (ab213524) at 1/1000 dilution
Lane 1: Wild-type A549 treated with 100 ng/ml IFN gamma (Recombinant Human Interferon gamma protein (Active) ab259377) for 48 h cell lysate at 20 µg
Lane 2: CD274 knockout A549 treated with 100 ng/ml IFN gamma (Recombinant Human Interferon gamma protein (Active) ab259377) for 48 h cell lysate at 20 µg
Lane 3: U-87 MG cell lysate at 20 µg
Lane 4: MCF7 cell lysate at 20 µg
Lane 5: Wild-type A549 untreated cell lysate at 20 µg
Lane 6: CD274 knockout A549 untreated cell lysate at 20 µg
Performed under reducing conditions.
Predicted band size: 33 kDa
Observed band size: 50 kDa
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PD-L1 antibody [EPR19759] (ab213524)
Immunohistochemical analysis of paraffin-embedded human placenta tissue labeling PD-L1 with ab213524 at 1/250 dilution, followed by Rabbit specific IHC polymer detection kit HRP/DAB (Rabbit specific IHC polymer detection kit HRP/DAB ab209101).
Membrane staining on the human placenta is observed.
Counter stained with Hematoxylin.
Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is rabbit specific IHC polymer detection kit HRP/DAB (Rabbit specific IHC polymer detection kit HRP/DAB ab209101)
Perform heat mediated antigen retrieval before commencing with IHC staining protocol.
Western blot - Anti-PD-L1 antibody [EPR19759] (ab213524)
Blocking/Dilution buffer: 5% NFDM/TBST.
The lower band is predicted to be isoform 2.
All lanes: Western blot - Anti-PD-L1 antibody [EPR19759] (ab213524) at 1/1000 dilution
Lane 1: Chinese hamster ovary cell lysate at 10 µg
Lane 2: Chinese hamster ovary cell lysate overexpressing PD-L1 at 10 µg
Secondary
All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/100000 dilution
Predicted band size: 33 kDa
Observed band size: 40-60 kDa
Exposure time: 3s
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PD-L1 antibody [EPR19759] (ab213524)
Tissue Microarrays stained for "Anti-PD-L1 antibody [EPR19759]" using "ab213524"in immunohistochemical analysis. This table provides a detailed overview of positive (tick mark) and negative (cross mark) staining per sample type tested. The sections were perform heat mediated antigen retrieval before commencing with IHC staining protocol. The sections were incubated with ab213524 at +4°C overnight. The secondary antibody is rabbit specific IHC polymer detection kit HRP/DAB (Rabbit specific IHC polymer detection kit HRP/DAB ab209101).
Western blot - Anti-PD-L1 antibody [EPR19759] (ab213524)
Anti-PD-L1 antibody [73-10] ab228415 works better than ab213524 in western blot testing.
We suggest optimizing experimental protocols (increasing lysate amount, using lower dilution or higher sensitivity ECL substrate) to improve results.
Blocking/Diluting buffer and concentration: 5% NFDM/TBST
All lanes: Western blot - Anti-PD-L1 antibody [EPR19759] (ab213524) at 1/1000 dilution
Lane 1: PC-3 (human prostate adenocarcinoma epithelial cell) whole cell lysate at 20 µg
Lane 2: A375 (human malignant melanoma epithelial cell) whole cell lysate at 20 µg
Lane 3: HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate at 20 µg
Lane 4: A549 (human lung carcinoma epithelial cell) whole cell lysate at 20 µg
Secondary
All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/20000 dilution
Observed band size: 40-60 kDa
Exposure time: 100s
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PD-L1 antibody [EPR19759] (ab213524)
Immunohistochemical analysis of formalin fixed paraffin embedded Human tonsil labelling PD L1 with ab213524 at a concentration of 0.5 µg/ml. The immunostaining was performed on a Ventana DISCOVERY ULTRA (Roche Tissue Diagnostics) instrument with a OptiView DAB IHC Detection Kit. Heat mediated antigen retrieval was performed with DISCOVERY cell conditioning solution (CC1) 100°C, pH8.5 for 32mins.
ab213524 anti PD L1 antibody EPR19759 was incubated for 16mins at 37°C. Sections were counterstained with Hematoxylin II. Image inset shows absence of staining in secondary antibody only control.
Customers are encouraged to optimise antigen retrieval conditions, antibody concentration, incubation times and temperature for best results in their own IHC assay workflow (automated and manual)