CD274 KO cell lysate available now. KO validated by Western blot. Free of charge wild type control included. Knockout achieved by using CRISPR/Cas9, 1 bp insertion in exon4 and 2 bp deletion in exon4 and 7 bp deletion in exon4.
Images
Key facts
- Cell type
A549
- Species or organism
Human
- Tissue
Lung
- Knockout validation
Sanger Sequencing, Western blot
- Mutation description
Knockout achieved by using CRISPR/Cas9, 1 bp insertion in exon4 and 2 bp deletion in exon4 and 7 bp deletion in exon4.
Alternative names
B7 H, B7 homolog 1, B7-H1, CD-274, CD274 antigen, CD274 molecule, MGC142294, MGC142296, OTTHUMP00000021029, PD-L1, PD1L1_HUMAN, PDCD1 ligand 1, PDCD1L1, PDCD1LG1, Programmed cell death 1 ligand 1, Programmed death ligand 1, RGD1566211
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CD274 KO cell lysate available now. KO validated by Western blot. Free of charge wild type control included. Knockout achieved by using CRISPR/Cas9, 1 bp insertion in exon4 and 2 bp deletion in exon4 and 7 bp deletion in exon4.
Key facts
- Cell type
A549
- Species or organism
Human
- Tissue
Lung
- Knockout validation
Sanger Sequencing, Western blot
- Mutation description
Knockout achieved by using CRISPR/Cas9, 1 bp insertion in exon4 and 2 bp deletion in exon4 and 7 bp deletion in exon4.
- Disease
Carcinoma
- Concentration
Properties
- Gene name
CD274
- Gene editing type
Knockout
- Gene editing method
CRISPR technology
- Knockout validation
Sanger Sequencing, Western blot
Quality control
- STR analysis
CSF1PO, D13S317, D7S820, D5S818, TH01, D16S539, TPOX
Cell culture
- Biosafety level
EU: 1 US: 1
- Adherent/suspension
Adherent
- Gender
Male
Storage
- Shipped at conditions
Ambient - Can Ship with Ice
- Appropriate short-term storage conditions
-20°C
- Appropriate long-term storage conditions
-20°C
Notes
Knockout cell lysate achieved by CRISPR/Cas9.
Treatments:
Human CD274 knockout A549 cell lysate - Untreated
Human wild-type A549 cell lysate - Untreated
Human CD274 knockout A549 cell lysate - IFN gamma treated (100 ng/ml, 48h)
Human wild-type A549 cell lysate - IFN gamma treated (100 ng/ml, 48h)
Abcam has not and does not intend to apply for the REACH Authorisation of customers' uses of products that contain European Authorisation list (Annex XIV) substances.
It is the responsibility of our customers to check the necessity of application of REACH Authorisation, and any other relevant authorisations, for their intended uses.
Lysate preparation: Our lysates are made using RIPA buffer to which we add a protease inhibitor cocktail and phosphatase inhibitor cocktail (ratio: 300:100:10). This means that the protein of interest is denatured. If you require a native form of the protein please use the live cell version. Please refer to our lysis protocol for further details on how our lysates are prepared.
User storage instructions: Lyophilizate may be stored at 4°C. After reconstitution, store at -20°C for short-term storage or -80°C for long-term storage.
This product is subject to limited use licenses from The Broad Institute, ERS Genomics Limited and Sigma-Aldrich Co. LLC, and is developed with patented technology. For full details of the licenses and patents please refer to our limited use license and patent pages.
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.
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8 product images
Western blot - Human CD274 (PD-L1) knockout A549 cell lysate (ab256831)
Lane 1: Wild-type A549 Treated IFN-gamma (100 ng/ml) for 48 hours cell lysate 20 ugg
Lane 2: CD274 knockout A549 Treated IFN-gamma (100 ng/ml) for 48 hours cell lysate 20 uggFalse colour image of Western blot: Anti-PD-L1 antibody [CAL10] – Rat IgG2a (Chimeric) staining at 1/1000 dilution, shown in green; Rabbit anti-alpha Tubulin antibody [EP1332Y] (Anti-alpha Tubulin antibody [EP1332Y] - Microtubule Marker ab52866) loading control staining at 1/20000 dilution, shown in red. In Western blot, Anti-PD-L1 antibody [CAL10] - Rat IgG2a (Chimeric) 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 Human CD274 (PD-L1) knockout A549 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 (Goat Anti-Rabbit IgG H&L (IRDye® 680RD) preadsorbed ab216777) at 1/20000 dilution.
All lanes: Western blot - Anti-PD-L1 antibody [CAL10] - Rat IgG2a (Chimeric) (Anti-PD-L1 antibody [CAL10] - Rat IgG2a (Chimeric) ab279294) at 1/1000 dilution
Lane 1: Wild-type A549 Treated IFN-gamma (100 ng/ml) for 48 hours cell lysate at 20 µg
Lane 2: CD274 knockout A549 Treated IFN-gamma (100 ng/ml) for 48 hours cell lysate at 20 µg
Performed under reducing conditions.
Predicted band size: 33 kDa
Observed band size: 48 kDa
Western blot - Human CD274 (PD-L1) knockout A549 cell lysate (ab256831)
Lane 1: Wild-type A549 Treated IFN-gamma (100 ng/ml) for 48 hours cell lysate 20 ugg
Lane 2: CD274 knockout A549 Treated IFN-gamma (100 ng/ml) for 48 hours cell lysate 20 ugg
False colour image of Western blot: Anti-PD-L1 antibody [CAL10] - Mouse IgG1 staining at 1/1000 dilution, shown in green; Rabbit anti-alpha Tubulin antibody [EP1332Y] (Anti-alpha Tubulin antibody [EP1332Y] - Microtubule Marker ab52866) loading control staining at 1/20000 dilution, shown in red. In Western blot, Anti-PD-L1 antibody [CAL10] - Mouse IgG1 (Chimeric) ab279292 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 Human CD274 (PD-L1) knockout A549 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-Mouse IgG H&L (IRDye® 800CW) preabsorbed (Goat anti-Mouse IgG H&L (IRDye® 800CW) preadsorbed ab216772) and Goat anti-Rabbit IgG H&L (IRDye® 680RD) preabsorbed (Goat Anti-Rabbit IgG H&L (IRDye® 680RD) preadsorbed ab216777) at 1/20000 dilution.Lanes 1 - 2: Western blot - Anti-PD-L1 antibody [CAL10] - Mouse IgG1 (Chimeric) (Anti-PD-L1 antibody [CAL10] - Mouse IgG1 (Chimeric) ab279292) at 1/1000 dilution
Lanes 1 - 2: Western blot - Anti-PD-L1 antibody [CAL10] (Anti-PD-L1 antibody [CAL10] ab237726) at 1/1000 dilution
Lane 1: Wild-type A549 Treated IFN-gamma (100 ng/ml) for 48 hours cell lysate at 20 µg
Lane 2: CD274 knockout A549 Treated IFN-gamma (100 ng/ml) for 48 hours cell lysate at 20 µg
Performed under reducing conditions.
Predicted band size: 33 kDa
Observed band size: 48 kDa
Western blot - Human CD274 (PD-L1) knockout A549 cell lysate (ab256831)
Lane 1: Wild-type A549 treated with 100 ng/mL IFN gamma (Recombinant Human Interferon gamma protein (Active) ab259377) for 48 h cell lysate (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 (20 μg)
Lane 3: U-87 MG cell lysate (20 μg)
Lane 4: MCF7 cell lysate (20 μg)
Lane 5: Wild-type A549 untreated cell lysate (20 μg)
Lane 6: CD274 knockout A549 untreated cell lysate (20 μg)
Lanes 1 - 6: Merged signal (red and green). Green - Anti-PD-L1 antibody [EPR19759] ab213524 observed at 50 kDa. Red - loading control, Anti-GAPDH antibody [6C5] - Loading Control ab8245 observed at 37 kDa.
Anti-PD-L1 antibody [EPR19759] 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 ab267054 (treated and untreated knockout cell lysates ab256831) 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. Anti-PD-L1 antibody [EPR19759] 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] (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
Western blot - Human CD274 (PD-L1) knockout A549 cell lysate (ab256831)
All lanes: Western blot - Anti-PD-L1 antibody [CAL10] - Mouse IgG2a (Chimeric) (Anti-PD-L1 antibody [CAL10] - Mouse IgG2a (Chimeric) ab279293) at 1/1000 dilution
Lane 1: Wild-type A549 Treated IFN-gamma (100 ng/mL, 48 h) cell lysate at 20 µg
Lane 2: Wild-type A549 Vehicle Control IFN-gamma (0 ng/mL, 48 h) cell lysate at 20 µg
Lane 3: CD274 knockout A549 Treated IFN-gamma (100 ng/mL, 48 h) cell lysate at 20 µg
Lane 4: CD274 knockout A549 Vehicle Control IFN-gamma (0 ng/mL, 48 h) cell lysate at 20 µg
Lane 5: Human Placenta cell lysate at 20 µg
SecondaryLanes 1 - 5: Goat anti-Mouse IgG H&L 800CW at 1/20000 dilution
Lanes 1 - 5: Goat anti-Rabbit IgG H&L 680RD at 1/20000 dilution
Performed under reducing conditions.
Observed band size: 45-65 kDa
Sanger Sequencing - Human CD274 (PD-L1) knockout A549 cell lysate (ab256831)
Allele-2: 2 bp deletion in exon4
Sanger Sequencing - Human CD274 (PD-L1) knockout A549 cell lysate (ab256831)
Allele-1: 7 bp deletion in exon4
Western blot - Human CD274 (PD-L1) knockout A549 cell lysate (ab256831)
Western blot: Anti-CD274 antibody [73-10] (Anti-PD-L1 antibody [73-10] ab228415) staining at 1/1000 dilution, shown in green; Mouse anti-Alpha Tubulin [DM1A] (Anti-alpha Tubulin antibody [DM1A] - Loading Control ab7291) loading control staining at 1/20000 dilution, shown in magenta. In Western blot, Anti-PD-L1 antibody [73-10] ab228415 was shown to bind specifically to CD274. A band was observed at 40-60 kDa in treated wild-type A549 cell lysates with no signal observed at this size in CD274 knockout cell line Human CD274 (PD-L1) knockout A549 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-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution.
All lanes: Western blot - Anti-PD-L1 antibody [73-10] (Anti-PD-L1 antibody [73-10] ab228415) at 1/1000 dilution
Lane 1: Wild-type A549 Treated IFN-gamma (100 ng/mL, 48 h) cell lysate at 20 µg
Lane 2: Wild-type A549 Vehicle Control IFN-gamma (0 ng/mL, 48 h) cell lysate at 20 µg
Lane 3: CD274 knockout A549 Treated IFN-gamma (100 ng/mL, 48 h) cell lysate at 20 µg
Lane 4: CD274 knockout A549 Vehicle Control IFN-gamma (0 ng/mL, 48 h) cell lysate at 20 µg
SecondaryAll lanes: Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution
Performed under reducing conditions.
Sanger Sequencing - Human CD274 (PD-L1) knockout A549 cell lysate (ab256831)
Allele-3: 1 bp insertion in exon4
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