Recombinant Anti-PD-L1 antibody [RM1012] (ab282458)
Key features and details
- Produced recombinantly (animal-free) for high batch-to-batch consistency and long term security of supply
- Rabbit recombinant multiclonal [RM1012] to PD-L1
- Suitable for: Flow Cyt, IP, Indirect ELISA, ICC/IF, WB, IHC-P
- Knockout validated
- Reacts with: Human
Related conjugates and formulations
Overview
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Product name
Anti-PD-L1 antibody [RM1012]
See all PD-L1 primary antibodies -
Description
Rabbit recombinant multiclonal [RM1012] to PD-L1 -
Host species
Rabbit -
Tested applications
Suitable for: Flow Cyt, IP, Indirect ELISA, ICC/IF, WB, IHC-Pmore details -
Species reactivity
Reacts with: Human -
Immunogen
Recombinant fragment. This information is proprietary to Abcam and/or its suppliers.
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Positive control
- WB: NCI-H1975 and MDA-MB-231 whole cell lysates; Human placenta tissue lysate, Human lung carcinoma epithelial cell) cell lysate, CD274 (PD-L1) KO A549, wild-type A549 cell lysate, Wild-type A549, MDA-MB-231 (Human breast adenocarcinoma epithelial cell) cell lysate, NCI-H1975 (Human adenocarcinoma lung epithelial cell) cell lysate IHC-P: Human breast cancer, lung cancer, placenta and tonsil tissue. ICC/IF & Flow Cyt: CHO-PDL1 cells. IP: NCI-H1975 whole cell lysates.
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General notes
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 production
Our RabMAb® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMAb® patents.
Properties
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Form
Liquid -
Storage instructions
Shipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C. Avoid freeze / thaw cycle. -
Storage buffer
pH: 7.2
Preservative: 0.01% Sodium azide
Constituents: 59.94% PBS, 40% Glycerol (glycerin, glycerine), 0.05% BSA -
Concentration information loading...
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Purity
Protein A purified -
Clonality
Recombinant Multiclonal -
Clone number
RM1012 -
Isotype
IgG -
Research areas
Associated products
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Compatible Secondaries
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Isotype control
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Related Products
Applications
The Abpromise guarantee
Our Abpromise guarantee covers the use of ab282458 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 |
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Flow Cyt |
1/50.
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IP |
1/30.
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Indirect ELISA |
Use a concentration of 0.25 µg/ml.
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ICC/IF |
1/1000.
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WB |
1/1000. Detects a band of approximately 40-60 kDa (predicted molecular weight: 33 kDa).
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IHC-P |
1/500. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.
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Notes |
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Flow Cyt
1/50. |
IP
1/30. |
Indirect ELISA
Use a concentration of 0.25 µg/ml. |
ICC/IF
1/1000. |
WB
1/1000. Detects a band of approximately 40-60 kDa (predicted molecular weight: 33 kDa). |
IHC-P
1/500. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol. |
Target
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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. - Information by UniProt
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Database links
- Entrez Gene: 29126 Human
- Omim: 605402 Human
- SwissProt: Q9NZQ7 Human
- Unigene: 521989 Human
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Alternative names
- B7 H antibody
- B7 H1 antibody
- B7 homolog 1 antibody
see all
Images
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All lanes : Anti-PD-L1 antibody [RM1012] (ab282458) at 1/10000 dilution
Lane 1 : Untreated CD274 (PD-L1) KO A549 (Human lung carcinoma epithelial cell) cell lysate with Intercept® (TBS) Blocking Buffer diluted with an equal volume of 0.1% TBS
Lane 2 : CD274 (PD-L1) KO A549 treated with 100 ng/ml human IFN gamma for 48 hours cell lysate with Intercept® (TBS) Blocking Buffer diluted with an equal volume of 0.1% TBS
Lane 3 : Untreated wild-type A549 cell lysate with Intercept® (TBS) Blocking Buffer diluted with an equal volume of 0.1% TBS
Lane 4 : Wild-type A549 treated with 100ng/ml human IFN gamma for 48 hours cell lysate with Intercept® (TBS) Blocking Buffer diluted with an equal volume of 0.1% TBS
Lane 5 : MDA-MB-231 (Human breast adenocarcinoma epithelial cell) cell lysate with Intercept® (TBS) Blocking Buffer diluted with an equal volume of 0.1% TBS
Lane 6 : NCI-H1975 (Human adenocarcinoma lung epithelial cell) cell lysate with Intercept® (TBS) Blocking Buffer diluted with an equal volume of 0.1% TBS
Lysates/proteins at 20 µg per lane.
Secondary
All lanes : Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) at 1/100000 dilution
Performed under reducing conditions.
Predicted band size: 33 kDa
Observed band size: 40-60 kDa why is the actual band size different from the predicted?
Exposure time: 3 minutesLysates/proteins at 20 µg per lane.
Performed under reducing conditions.
False colour image of Western blot: Anti-PD-L1 antibody [RM1012] (ab282458) staining at 1/10, 000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in red.
In Western blot, ab282458 was shown to bind specifically to PD-L1. Target band was observed at 40-60 kDa in wild-type A549 treated with IFN gamma cell lysates with no signal observed at this size in PD-L1 knockout cell line ab267054 (knockout cell lysate ab256831). To generate this image, wild-type and CD274 (PD-L1) knockout A549 cell lysates were analyzed. 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-Rabbit IgG H&L (IRDye® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed (ab216776) at 1/20000 dilution.
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Immunohistochemical analysis of paraffin-embedded Human lung cancer tissue labelling PD-L1 with ab282458 at 1/500 (0.876 ug/ml) dilution followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Positive staining on human lung cancer. The section was incubated with ab282458 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Counterstained with Hematoxylin.
Secondary antibody only control: Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).
Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins
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Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized CHO-PDL1 cells labelling PD-L1 with ab282458 at 1/1000 (0.438 ug/ml) dilution, followed by ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) antibody at 1/1000 dilution (Green). Confocal image showing membranous staining in CHO-PDL1 cells and no staining in CHO-S cells.
ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 2.5ug/ml dilution (Red). The Nuclear counterstain was DAPI (Blue).
Secondary antibody only control: Secondary antibody is ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) at 1/1000 dilution.
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All lanes : Anti-PD-L1 antibody [RM1012] (ab282458) at 1/1000 dilution
Lane 1 : NCI-H1975 (Human adenocarcinoma lung epithelial cell) whole cell lysate
Lane 2 : MDA-MB-231 (Human breast adenocarcinoma epithelial cell) whole cell lysate
Lane 3 : MCF7 (Human breast adenocarcinoma epithelial cell) whole cell lysate
Lane 4 : Human placenta lysate
Lysates/proteins at 20 µg per lane.
Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution (Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated)
Predicted band size: 33 kDa
Observed band size: 40-60 kDa why is the actual band size different from the predicted?Blocking and Diluting buffer and concentration: 5% NFDM/TBST.
Low expression control: MCF7 (PMID: 28184013, 31741201).
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Flow cytometric analysis of CHO-S (Chinese hamster ovary epithelial cell (Blue)) / CHO-PDL1 (PD-L1 stably expessed Chinese hamster ovary epithelial cell) (Red)) cells labelling PD-L1 with ab282458 at 1/50 dilution (1ug). Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) at 1/2000 dilution was used as the secondary antibody. Gated on viable cells.
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PD-L1 was immunoprecipitated from 0.35 mg NCI-H1975 (Human adenocarcinoma lung epithelial cell) whole cell lysate with ab282458 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab282458 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution.
Lane 1: NCI-H1975 (Human adenocarcinoma lung epithelial cell) whole cell lysate 10 ug
Lane 2: ab282458 IP in NCI-H1975 whole cell lysate
Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab282458 in NCI-H1975 whole cell lysate
Blocking and dilution buffer and concentration: 5% NFDM/TBST.
Exposure time: 32 seconds
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Immunohistochemical analysis of paraffin-embedded Human breast cancer tissue labelling PD-L1 with ab282458 at 1/500 (0.876 ug/ml) dilution followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Positive staining on human breast cancer. The section was incubated with ab282458 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Counterstained with Hematoxylin.
Secondary antibody only control: Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).
Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins
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ELISA using ab282458 at varying antibody concentrations and antigen concentration at 1000 ng/ml. An Alkaline Phosphatase-conjugated AffiniPure Goat Anti-Rabbit IgG (H+L) (1/2500) was used as the secondary antibody.
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Immunohistochemical analysis of paraffin-embedded Human placenta tissue labelling PD-L1 with ab282458 at 1/500 (0.876 ug/ml) dilution followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Positive staining on human placenta. The section was incubated with ab282458 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Counterstained with Hematoxylin.
Secondary antibody only control: Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).
Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins
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Immunohistochemical analysis of paraffin-embedded Human tonsil tissue labelling PD-L1 with ab282458 at 1/500 (0.876 ug/ml) dilution followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Positive staining on human tonsil. The section was incubated with ab282458 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Counterstained with Hematoxylin.
Secondary antibody only control: Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).
Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins
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.
Datasheets and documents
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SDS download
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Datasheet download
References (1)
ab282458 has been referenced in 1 publication.
- Deng X et al. Upregulation of MTHFD2 is associated with PD‑L1 activation in bladder cancer via the PI3K/AKT pathway. Int J Mol Med 51:N/A (2023). PubMed: 36601741