Human CD274 (PD-L1) knockout A549 cell lysate (ab256831)
Overview
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Product name
Human CD274 (PD-L1) knockout A549 cell lysate
See all PD-L1 kits -
Product overview
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) -
Parental Cell Line
A549 -
Organism
Human -
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. -
Passage number
<20 -
Knockout validation
Sanger Sequencing, Western Blot (WB) -
Reconstitution notes
To use as WB control, resuspend the lyophilizate in 50 µL of LDS* Sample Buffer to have a final concentration of 2 mg/ml. For reducing conditions, we recommend a final concentration of 0.1 M DTT.*Usage of SDS sample buffer is not recommended with these lyophilized lysates.
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Notes
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 - found here. 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.
Access thousands of knockout cell lysates, generated from commonly used cancer cell lines.
See here for more information on knockout cell lysates.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.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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Tested applications
Suitable for: WBmore details
Properties
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Storage instructions
Store at -80°C. Please refer to protocols. -
Components 1 kit ab277651 - Human CD274 knockout A549 cell lysate - IFN gamma treated 1 x 100µg ab263528 - Human CD274 knockout A549 cell lysate 1 x 100µg ab277652 - Human wild-type A549 cell lysate - IFN gamma treated 1 x 100µg ab255554 - Human wild-type A549 cell lysate 1 x 100µg -
Research areas
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Cell type
epithelial -
Disease
Carcinoma -
STR Analysis
Amelogenin X,Y D5S818: 11 D13S317: 11 D7S820: 8, 11 D16S539: 11, 12 vWA: 14 TH01: 8,9.3 TPOX: 8,11 CSF1PO: 10, 12
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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Alternative names
- B7 H
- B7 H1
- B7 homolog 1
see all
Associated products
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KO cell lines
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Recombinant Protein
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Related Products
Applications
The Abpromise guarantee
Our Abpromise guarantee covers the use of ab256831 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 |
|---|---|---|
| WB |
Use at an assay dependent concentration. Predicted molecular weight: 33 kDa.
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| Notes |
|---|
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WB
Use at an assay dependent concentration. Predicted molecular weight: 33 kDa. |
Images
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Western blot - Human CD274 (PD-L1) knockout A549 cell lysate (ab256831)
Lane 1: Wild-type A549 Treated IFN-gamma (100 ng/mL, 48 h) cell lysate 20 μg
Lane 2: Wild-type A549 Vehicle Control IFN-gamma (0 ng/mL, 48 h) cell lysate 20 μg
Lane 3: CD274 knockout A549 Treated IFN-gamma (100 ng/mL, 48 h) cell lysate 20 μg
Lane 4: CD274 knockout A549 Vehicle Control IFN-gamma (0 ng/mL, 48 h) cell lysate 20 μg
Lane 5: Human Placenta cell lysate 20 μgFalse colour image of Western blot: Anti-PD-L1 antibody [CAL10] - Mouse IgG2a staining at 1/1000 dilution, shown in green; Rabbit Anti-GAPDH antibody [EPR16891] (ab181602) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab279293 was shown to bind specifically to PD-L1. A band was observed at 45-65 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 fluorescent western blot (TBS-based) blocking solution 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 800CW and Goat anti-Rabbit IgG H&L 680RD at 1/20000 dilution.
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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 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 - 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. -
Western blot - Human CD274 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. -
Sanger Sequencing - Human CD274 knockout A549 cell lysate (ab256831)Allele-1: 7 bp deletion in exon4
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Sanger Sequencing - Human CD274 knockout A549 cell lysate (ab256831)Allele-2: 2 bp deletion in exon4
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Sanger Sequencing - Human CD274 knockout A549 cell lysate (ab256831)Allele-3: 1 bp insertion in exon4
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 (0)
ab256831 has not yet been referenced specifically in any publications.