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Antibody Identity card

Antibody information [ PDL-1 mouse specific]

EPR20529

  • Clone name EPR20529
  • Description Rabbit monoclonal
  • Antigen used Recombinant fragment
  • Epitope Unknown
  • Isotype IgG
  • Confirmed species reactivity Mouse
  • Ab used Abcam Anti-PD-L1 (ab213480) 0.508mg/ml
APPLICATIONRecommended concentrationStatusProtocol
Western blotting (WB)1 ug/mlWorkingOdyssey Western Blotting protocol (OdWB)
Immunocytochemistry (ICC)0.25 ug/mlWorkingICC in frozen tissue and cytospin preparation
Immunohistochemistry (IHC-P)0.25 ug/mlWorkingPDL-1 mAb IHC-P in mouse tissues
Immunoflourescence (IF)Not testedImmunofluorescence staining
Flow cytometry (FC)1.016 ug/mlWorkingFlow cytometry
IHC-P SpeciesNot tested

Ab ID Card application validation / characterisation

WB Validation
Over-expression/cross reactivity
PDL-1 mouse specific antibody WB Validation: Over-expression/cross reactivity
To confirm the lack of cross-reactivity with human PDL1, western blotting (WB) of the EPR20529 mAb was performed using cell extracts of mouse PDL1 (mPDL1), human PDL1 (hPDL1) and human CL7 HEK293T transfectants. A specific band of 48 kDa was detected by WB only in the HEK-mPDL1 cell extracts. No expression was found in the HEK-human PDL1 and hCL7 extracts (negative control). Anti-vinculin was used as loading control.

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Gene inactivation
PDL-1 mouse specific antibody WB Validation: Gene inactivation
Western blotting of the EPR20529 mAb using cell extracts of MEFs before (WT) and after activation with interferon gamma (IFNγ, 48h 150ng/ml) and PDL1 KO transfected MEFs after before and after IFNγ activation. As expected a specific 48kDa band is observed in only in the MEFs cell extract after IFNγ treatment. No bands are observed in the MEFs WT and MEFs mPDL1 KO with and without IFNγ treatment. Anti-vinculin was used as loading control.

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WB Characterisation
Endogenous expression
PDL-1 mouse specific antibody WB Characterisation: Endogenous expression
Western Blotting of the EPR20529 mAb illustrating PD-L1 and EGFP expression in different organs of PD-L1AT/+ mice and the spleen of PD-L1+/+ and PD-L1AT/AT mice. β-actin and GAPDH mAbs were used as loading controls. Image kindly donated by Dr. Elena Fueyo

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ICC / ICC-P Validation
Over-expression/cross reactivity
PDL-1 mouse specific antibody ICC Validation: Over-expression/cross reactivity
Immunocytochemistry (ICC) of the EPR20529 mAb was performed on frozen cytospin preparations of HEK293T cells transfected with MYC-tagged mouse PDL1 or PILRA proteins. Staining was observed only in the PDL-1 transfectants. Labelling with anti-MYC was used to confirm the efficiency of transfection.

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Gene inactivation
PDL-1 mouse specific antibody ICC Validation: Gene inactivation
Immunohistochemistry using the EPR20529 mAb on paraffin-embedded sections of mouse wild type (WT) and mPDL1 knock out (KO) lymph nodes. Membrane staining on dendritic cells is present in the WT lymph node. No staining is found in the mPDL1 KO lymph node.

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IHC-P Characterisation
Endogenous expression
PDL-1 mouse specific antibody IHC-P Characterisation: Endogenous expression
Immunohistochemistry on paraffin embedded sections of mouse lymph node, spleen, lung, placenta, kidney and testicle using the EPR20529 mAb.

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IF Characterisation
Endogenous expression
PDL-1 mouse specific antibody IF Characterisation: Endogenous expression
Double immunofluorescence staining of EGFP (green) and PD-L1 (clone EPR20529) (red) in PD-L1AT/+ MEFs exposed to IFNγ (50 ng/ml) for 24 hours. Scale bar (white) indicates 5 μm. Image kindly donated by Dr. Elena Fueyo

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FC Validation
Over-expression/cross reactivity
PDL-1 mouse specific antibody FC Validation: Over-expression/cross reactivity
Overexpression of mPDL1 protein was tested using the EPR20529 mAb in flow cytometry. HEK293T cells transfected with mPDL1 were used as a positive control (light blue) while HEK293T cell transfected with TOX2 were the negative control (red).

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Gene inactivation
PDL-1 mouse specific antibody FC Validation: Gene inactivation
Flow cytometry using wild type MEFs (WT MEFs) before (blue) and after activation with interferon gamma (IFNγ, 48h 150ng/ml)(red), PD-L1 knockouts before (PD-L1 KO MEFs, green) and after activation with IFNγ (48h 150ng/ml, orange). Image kindly donated by Dr. Elena Fueyo

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FC Characterisation
Endogenous expression
Not tested
IHC-P Domestic species
Not tested
IHC-P Wild species
Not tested
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