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    products/primary-antibodies/pd-l1-antibody-cal10-rat-igg2a-ab279294.pdf

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Immunology Cell Type Markers CD Non-lineage
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Validated using a knockout cell lineRecombinant

Recombinant Anti-PD-L1 antibody [CAL10] - Rat IgG2a (ab279294)

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Western blot - Anti-PD-L1 antibody [CAL10] - Rat IgG2a (ab279294)
  • Western blot - Anti-PD-L1 antibody [CAL10] (ab279294)
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PD-L1 antibody [CAL10] (ab279294)
  • Immunocytochemistry/ Immunofluorescence - Anti-PD-L1 antibody [CAL10] - Rat IgG2a (ab279294)
  • Flow Cytometry (Intracellular) - Anti-PD-L1 antibody (ab279294)

Key features and details

  • Produced recombinantly (animal-free) for high batch-to-batch consistency and long term security of supply
  • Rat monoclonal [CAL10] to PD-L1 - Rat IgG2a
  • Suitable for: Flow Cyt (Intra), IHC-P, ICC/IF, WB
  • Knockout validated
  • Reacts with: Human

Conjugates logo Related conjugates and formulations

Alexa Fluor® 555 Carrier Free

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Overview

  • Product name

    Anti-PD-L1 antibody [CAL10] - Rat IgG2a
    See all PD-L1 primary antibodies
  • Description

    Rat monoclonal [CAL10] to PD-L1 - Rat IgG2a
  • Host species

    Rat
  • Tested applications

    Suitable for: Flow Cyt (Intra), IHC-P, ICC/IF, WBmore details
  • Species reactivity

    Reacts with: Human
  • Immunogen

    Synthetic peptide. This information is proprietary to Abcam and/or its suppliers.

  • Positive control

    • WB: PD-L1 stably expressed CHO whole cell lysate. Human placenta tissue lysate. NCI-H1299 whole cell lysate. ICC/IF: PD-L1 stably expressed CHO cells. Flow Cyt (intra): PD-L1 stably expressed CHO cells. IHC-P: Human tonsil tissue.
  • General notes

    This rat antibody has been engineered from a RabMAb parent antibody (ab237726). By necessity, some rabbit sequence is retained as part of the variable domain. When multiplexing with other rabbit-derived antibodies, using cross absorbed Fc-reactive secondary antibodies are recommended.

Properties

  • Form

    Liquid
  • Storage instructions

    Shipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C long term. Avoid freeze / thaw cycle.
  • Storage buffer

    pH: 7.2
    Preservative: 0.01% Sodium azide
    Constituents: 59% PBS, 40% Glycerol (glycerin, glycerine), 0.05% BSA
  • Concentration information loading...
  • Purity

    Protein A purified
  • Clonality

    Monoclonal
  • Clone number

    CAL10
  • Isotype

    IgG2a
  • Research areas

    • Immunology
    • Cell Type Markers
    • CD
    • Non-lineage
    • Immunology
    • Immune System Diseases
    • Autoimmune
    • Cancer
    • Tumor immunology
    • Tumor-associated antigens
    • Cancer
    • Tumor biomarkers
    • Tumor antigens

Associated products

  • Alternative Versions

    • Anti-PD-L1 antibody [CAL10] - Chimeric – BSA and Azide free (ab279306)
  • Compatible Secondaries

    • Rabbit Anti-Rat IgG H&L preadsorbed (ab102248)
    • Goat Anti-Rat IgG H&L (Alexa Fluor® 488) (ab150157)
    • Goat F(ab')2 Anti-Rat IgG Fc (Alexa Fluor® 488) preadsorbed (ab150161)
    • Goat Anti-Rat IgG H&L (HRP) (ab205720)
  • Related Products

    • Goat Anti-Rabbit IgG H&L (Alexa Fluor® 594) (ab150080)
    • Anti-beta Tubulin antibody [EPR16774] (ab179513)

Applications

The Abpromise guarantee

Our Abpromise guarantee covers the use of ab279294 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

Application Abreviews Notes
Flow Cyt (Intra)
1/50.
IHC-P
Use a concentration of 1 µg/ml. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.
ICC/IF
1/100.
WB
1/1000.
Notes
Flow Cyt (Intra)
1/50.
IHC-P
Use a concentration of 1 µg/ml. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.
ICC/IF
1/100.
WB
1/1000.

Target

  • Function

    Involved in the costimulatory signal, essential for T-cell proliferation and production of IL10 and IFNG, in an IL2-dependent and a PDCD1-independent manner. Interaction with PDCD1 inhibits T-cell proliferation and cytokine production.
  • Tissue specificity

    Highly expressed in the heart, skeletal muscle, placenta and lung. Weakly expressed in the thymus, spleen, kidney and liver. Expressed on activated T- and B-cells, dendritic cells, keratinocytes and monocytes.
  • Sequence similarities

    Belongs to the immunoglobulin superfamily. BTN/MOG family.
    Contains 1 Ig-like C2-type (immunoglobulin-like) domain.
    Contains 1 Ig-like V-type (immunoglobulin-like) domain.
  • Cellular localization

    Cell membrane and Endomembrane system.
  • Target information above from: UniProt accession Q9NZQ7 The UniProt Consortium
    The Universal Protein Resource (UniProt) in 2010
    Nucleic Acids Res. 38:D142-D148 (2010) .

    Information by UniProt
  • Database links

    • Entrez Gene: 29126 Human
    • Omim: 605402 Human
    • SwissProt: Q9NZQ7 Human
    • Unigene: 521989 Human
    • Alternative names

      • B7 H antibody
      • B7 H1 antibody
      • B7 homolog 1 antibody
      • B7-H1 antibody
      • B7H antibody
      • B7H1 antibody
      • CD 274 antibody
      • CD-274 antibody
      • CD274 antibody
      • CD274 antigen antibody
      • CD274 molecule antibody
      • MGC142294 antibody
      • MGC142296 antibody
      • OTTHUMP00000021029 antibody
      • PD L1 antibody
      • PD-L1 antibody
      • PD1L1_HUMAN antibody
      • PDCD1 ligand 1 antibody
      • PDCD1L1 antibody
      • PDCD1LG1 antibody
      • PDL 1 antibody
      • PDL1 antibody
      • Programmed cell death 1 ligand 1 antibody
      • Programmed death ligand 1 antibody
      • RGD1566211 antibody
      see all

    Images

    • Western blot - Anti-PD-L1 antibody [CAL10] - Rat IgG2a (ab279294)
      Western blot - Anti-PD-L1 antibody [CAL10] - Rat IgG2a (ab279294)
      All lanes : Anti-PD-L1 antibody [CAL10] - Rat IgG2a (ab279294) at 1/1000 dilution

      Lane 1 : Wild-type A549 Treated IFN-gamma (100 ng/ml) for 48 hours cell lysate
      Lane 2 : CD274 knockout A549 Treated IFN-gamma (100 ng/ml) for 48 hours cell lysate

      Lysates/proteins at 20 µg per lane.

      Performed under reducing conditions.

      Observed band size: 48 kDa why is the actual band size different from the predicted?



      False colour image of Western blot: Anti-PD-L1 antibody [CAL10] - Rat IgG2a staining at 1/1000 dilution, shown in green; Rabbit anti-alpha Tubulin antibody [EP1332Y] (ab52866) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab279294 was shown to bind specifically to PD-L1. A band was observed at 48 kDa in treated wild-type A549 cell lysates with no signal observed at this size in Cd274 knockout cell line ab267054 (knockout cell lysate ab256831). To generate this image, wild-type and Cd274 knockout A549 cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % milk in TBS-0.1 % Tween® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rat IgG H&L (IRDye® 800CW) preabsorbed (ab253031) and Goat anti-Rabbit IgG H&L (IRDye® 680RD) preabsorbed (ab216777) at 1/20000 dilution.

    • Western blot - Anti-PD-L1 antibody [CAL10] (ab279294)
      Western blot - Anti-PD-L1 antibody [CAL10] (ab279294)
      All lanes : Anti-PD-L1 antibody [CAL10] - Rat IgG2a (ab279294) at 1/1000 dilution

      Lane 1 : CHO-S (Chinese hamster ovary epithelial cell) whole cell lysate
      Lane 2 : CHO-PD-L1 (PD-L1 stably expressed Chinese hamster ovary epithelial cell) whole cell lysate
      Lane 3 : Human placenta tissue lysate
      Lane 4 : NCI-H1299 (human lung carcinoma epithelial cell), whole cell lysate

      Lysates/proteins at 20 µg per lane.

      Secondary
      All lanes : Goat Anti-Rat IgG H&L (HRP) (ab205720) at 1/5000 dilution

      Observed band size: 40-60 kDa why is the actual band size different from the predicted?


      Exposure time: 8 seconds


      Blocking/Dilution buffer: 5% NFDM/TBST.

    • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PD-L1 antibody [CAL10] (ab279294)
      Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PD-L1 antibody [CAL10] (ab279294)

      IHC image of PD-L1 staining in a section of formalin-fixed paraffin-embedded normal human tonsil* performed on a Leica BONDTM system using the standard protocol F.

      The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20mins. The section was then incubated with ab279294, 1ug/ml, for 15 mins at room temperature. A rabbit anti-rat IgG2a, ab102248, was added for 8 mins at room temperature and detected using an HRP conjugated goat anti-rabbit compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX. The inset secondary-only control image is taken from an identical assay without primary antibody.
      For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

      *Tissue obtained from the Human Research Tissue Bank, supported by the NIHR Cambridge Biomedical Research Centre

    • Immunocytochemistry/ Immunofluorescence - Anti-PD-L1 antibody [CAL10] - Rat IgG2a (ab279294)
      Immunocytochemistry/ Immunofluorescence - Anti-PD-L1 antibody [CAL10] - Rat IgG2a (ab279294)

      Immunocytochemical analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100-fixed permeabilized CHO-PD-L1 cells labeling PD-L1 with ab279294 at 1/100 dilution, followed by ab150157 Goat Anti-Rat IgG H&L (Alexa Fluor® 488) at 1/1000 dilution (Green). ab179513 Anti-beta Tubulin rabbit monoclonal antibody was used to counterstain tubulin at 1/200 dilution, followed by ab150080 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 594) at a 1/500 dilution (Red). The nuclear counterstain was DAPI (Blue). Confocal image showing membranous and cytoplasmic staining in CHO-PD-L1 cells.

      Negative control 1: ab279294 at a 1/100 dilution followed by ab150080 at a 1/200 dilution.

      Negative control 2: ab179513 at a 1/200 dilution followed by ab150157 at a 1/1000 dilution.

    • Flow Cytometry (Intracellular) - Anti-PD-L1 antibody (ab279294)
      Flow Cytometry (Intracellular) - Anti-PD-L1 antibody (ab279294)

      Flow cytometric analysis of 4% paraformaldehyde-fixed, 90% methanol-permeabilized CHO-s (Chinese hamster ovary epithelial cell, Blue) / CHO-PDL1 (PD-L1 stably expessed Chinese hamster ovary epithelial cell, Red) labelling PD-L1 with ab279294 at 1/50 dilution (0.1µg).

      Goat F(ab)2 Anti-Rat IgG Fc (Alexa Fluor® 488, ab150161) at 1/2000 dilution was used as the secondary antibody.

    Protocols

    To our knowledge, customised protocols are not required for this product. Please try the standard protocols listed below and let us know how you get on.

    Click here to view the general protocols

    Datasheets and documents

    • SDS download

    • Datasheet download

      Download

    References (0)

    Publishing research using ab279294? Please let us know so that we can cite the reference in this datasheet.

    ab279294 has not yet been referenced specifically in any publications.

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