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Antibody Identity card

Antibody information [ PDL-1 mouse specific]

GOYA536A

  • Clone name GOYA536A
  • Description Rat monoclonal
  • Antigen used RBL1 cell line transfected with full lenght mouse PDL1 vector
  • Epitope Unknown
  • Isotype IgG2a
  • Confirmed species reactivity Mouse
  • Ab used Rat mAb clone name GOYA536A CNIO (tissue culture
APPLICATIONRecommended concentrationStatusProtocol
Western blotting (WB)38μg/mlWorkingWestern Blotting (WB)
Immunocytochemistry (ICC)neat (tissue culture supernatant)WorkingCytospin preparation technique
Immunohistochemistry (IHC-P)Not tested
Immunoflourescence (IF)neat (tissue culture supernatant)Not testedImmunofluorescence staining
Flow cytometry (FC)38 μg/ml WorkingFlow 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 GOYA536A mAb was performed using cell extracts of HEK cells transfected with mouse PDL-1 (mPDL1), human PDL1 (hPDL1) and human CL7 (hCL7 as a negative control). A specific band of 48 kDa was detected by WB in HEK-mouse PDL1 cell extracts while no expression was found in the HEK-human PDL1 and hCL7 transfectants. 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 GOYA536A mAb using cell extracts of wild type (WT) MEFs before and after activation with interferon gamma (IFNγ, 150 ng/ml for 48 h), PDL1 knockout (KO) MEFs before and after IFNγ activation. As expected a specific band around 48 kDa is observed in the MEFs WT cell extract after interferon-gamma treatment. No bands are observed in MEFs WT without IFNγ treatment and in MEFs 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 GOYA536A mAb using cell extracts of wild type (WT) RAW264.7 before and after activation with interferon gamma (IFNγ, 150 ng/ml for 48 h). Bands were observed the RAW 264.7 extract after IFNγ. Anti-vinculin was used as loading control.

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ICC / ICC-P Validation
Over-expression/cross reactivity
PDL-1 mouse specific antibody ICC Validation: Over-expression/cross reactivity
To confirm the specificity of the GOYA536A mAb immunocytochemistry (ICC) was performed on frozen cytospin preparations of MYC-tagged murine PDL1 (mPDL1) and human PDL1 (hPDL1) proteins expressed in HEK293T cells. The GOYA536A mAb stained only the mPDL1 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 on parrafin sections of mouse wild type (WT) and mPDL1 knockout (mPDL1 KO) thymus using the GOYA536A mAb. Membrane labelling is present in the WT lymph node. No staining is found in mPDL1 KO thymus.

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IHC-P Characterisation
Endogenous expression
Not tested
IF Characterisation
Endogenous expression
PDL-1 mouse specific antibody IF Characterisation: Endogenous expression
Double immunofluorescence staining of EGFP (green) and PD-L1 (red) in PD-L1AT/+ mouse lung. Scale bar (white) indicates 30 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 GOYA536A 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 of the GOYA536A mAb using A) wild type (WT) MEFs before (blue) and after activation with interferon gamma (150ng/ml for 48 h, red) and B) knockout (KO) MEFs before (blue) and after IFNγ activation (150 ng/ml for 48 h, red). Image kindly donated by Dr. Elena Fueyo

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FC Characterisation
Endogenous expression
PDL-1 mouse specific antibody FC Characterisation: Endogenous expression
Flow cytometry of the GOYA536A mAb using the NS1 (orange), RAW264.7 (blue) and RAW264.7 cell lines after activation with interferon gamma (48h,150ng/ml)(red). Image kindly donated by Dr. Elena Fueyo

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IHC-P Domestic species
Not tested
IHC-P Wild species
Not tested
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