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AB256831

Human CD274 (PD-L1) knockout A549 cell lysate

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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.
8 Images
Western blot - Human CD274 (PD-L1) knockout A549 cell lysate (AB256831)
  • WB

Lab

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] (ab52866) loading control staining at 1/20000 dilution, shown in red. In Western blot, 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 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 (ab216772) and Goat anti-Rabbit IgG H&L (IRDye® 680RD) preabsorbed (ab216777) at 1/20000 dilution.

Lanes 1 - 2:

Western blot - Anti-PD-L1 antibody [CAL10] - Mouse IgG1 (Chimeric) (<a href='/en-us/products/primary-antibodies/pd-l1-antibody-cal10-mouse-igg1-chimeric-ab279292'>ab279292</a>) at 1/1000 dilution

Lanes 1 - 2:

Western blot - Anti-PD-L1 antibody [CAL10] (<a href='/en-us/products/primary-antibodies/pd-l1-antibody-cal10-ab237726'>ab237726</a>) 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

Lane 2:

Western blot - Human CD274 (PD-L1) knockout A549 cell line (<a href='/en-us/products/cell-lines/human-cd274-pd-l1-knockout-a549-cell-line-ab267054'>ab267054</a>)

Predicted band size: 33 kDa

Observed band size: 48 kDa

false

Western blot - Human CD274 (PD-L1) knockout A549 cell lysate (AB256831)
  • WB

Lab

Western blot - Human CD274 (PD-L1) knockout A549 cell lysate (AB256831)

All lanes:

Western blot - Anti-PD-L1 antibody [CAL10] - Mouse IgG2a (Chimeric) (<a href='/en-us/products/primary-antibodies/pd-l1-antibody-cal10-mouse-igg2a-chimeric-ab279293'>ab279293</a>) 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

Secondary

Lanes 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

Observed band size: 45-65 kDa

false

Western blot - Human CD274 (PD-L1) knockout A549 cell lysate (AB256831)
  • WB

Lab

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] – Rat IgG2a (Chimeric) 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.

All lanes:

Western blot - Anti-PD-L1 antibody [CAL10] - Rat IgG2a (Chimeric) (<a href='/en-us/products/primary-antibodies/pd-l1-antibody-cal10-rat-igg2a-chimeric-ab279294'>ab279294</a>) 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

Lane 2:

Western blot - Human CD274 (PD-L1) knockout A549 cell line (<a href='/en-us/products/cell-lines/human-cd274-pd-l1-knockout-a549-cell-line-ab267054'>ab267054</a>)

Predicted band size: 33 kDa

Observed band size: 48 kDa

false

Western blot - Human CD274 (PD-L1) knockout A549 cell lysate (AB256831)
  • WB

Lab

Western blot - Human CD274 (PD-L1) knockout A549 cell lysate (AB256831)

Western blot : Anti-CD274 antibody [73-10] (ab228415) staining at 1/1000 dilution, shown in green; Mouse anti-Alpha Tubulin [DM1A] (ab7291) loading control staining at 1/20000 dilution, shown in magenta. In Western blot, 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 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] (<a href='/en-us/products/primary-antibodies/pd-l1-antibody-73-10-ab228415'>ab228415</a>) 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

Secondary

All lanes:

Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution

false

Western blot - Human CD274 (PD-L1) knockout A549 cell lysate (AB256831)
  • WB

Lab

Western blot - Human CD274 (PD-L1) knockout A549 cell lysate (AB256831)

Lane 1 : Wild-type A549 treated with 100 ng/mL IFN gamma (ab259377) for 48 h cell lysate (20 μg)

Lane 2 : CD274 knockout A549 treated with 100 ng/mL IFN gamma (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 - ab213524 observed at 50 kDa. Red - loading control, ab8245 observed at 37 kDa.

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 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. ab213524 and 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 (ab216773) and 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] (<a href='/en-us/products/primary-antibodies/pd-l1-antibody-epr19759-ab213524'>ab213524</a>) at 1/1000 dilution

Lane 1:

Wild-type A549 treated with 100 ng/ml IFN gamma (<a href='/en-us/products/proteins-peptides/recombinant-human-interferon-gamma-protein-active-ab259377'>ab259377</a>) for 48 h cell lysate at 20 µg

Lane 2:

CD274 knockout A549 treated with 100 ng/ml IFN gamma (<a href='/en-us/products/proteins-peptides/recombinant-human-interferon-gamma-protein-active-ab259377'>ab259377</a>) 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

Predicted band size: 33 kDa

Observed band size: 50 kDa

false

Sanger Sequencing - Human CD274 (PD-L1) knockout A549 cell lysate (AB256831)
  • Sanger seq

Unknown

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)
  • Sanger seq

Unknown

Sanger Sequencing - Human CD274 (PD-L1) knockout A549 cell lysate (AB256831)

Allele-1 : 7 bp deletion in exon4

Sanger Sequencing - Human CD274 (PD-L1) knockout A549 cell lysate (AB256831)
  • Sanger seq

Unknown

Sanger Sequencing - Human CD274 (PD-L1) knockout A549 cell lysate (AB256831)

Allele-3 : 1 bp insertion 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

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Cell Lines & Lysates

AB267054

Human CD274 (PD-L1) knockout A549 cell line

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Western blot - Human CD274 (PD-L1) knockout A549 cell line (AB267054)

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Primary Antibodies

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Proteins & Peptides

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Reactivity data

{ "title": "Reactivity Data", "filters": { "stats": ["", "Reactivity", "Dilution Info", "Notes"] }, "values": { "WB": { "reactivity":"TESTED_AND_REACTS", "dilution-info":"", "notes":"<p></p>" } } }
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Product details

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)

REACH authorisation
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.

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What's included?

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Properties and storage information

Gene name
CD274
Gene editing type
Knockout
Gene editing method
CRISPR technology
Knockout validation
Sanger Sequencing, Western blot
Shipped at conditions
Ambient - Can Ship with Ice
Appropriate short-term storage conditions
-20°C
Appropriate long-term storage conditions
-20°C
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Supplementary information

This supplementary information is collated from multiple sources and compiled automatically.

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.

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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Quality control

STR analysis

CSF1PO, D13S317, D7S820, D5S818, TH01, D16S539, TPOX

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Cell culture

Biosafety level

EU: 1 US: 1

Adherent/suspension

Adherent

Gender

Male

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Product protocols

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Product promise

We are committed to supporting your work with high-quality reagents, and we're here for you every step of the way. In the unlikely event that one of our products does not perform as expected, you're protected by our Product Promise.
For full details, please see our Terms & Conditions

Please note: All products are 'FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC OR THERAPEUTIC PROCEDURES'.

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