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

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PD-L1 antibody [CAL10] - Rat IgG2a (Chimeric) (AB279294)

IHC image of PD-L1 staining in a section of formalin-fixed paraffin-embedded normal human tonsil* performed on a Leica BOND^TM 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

• WB

Lab

Western blot - Anti-PD-L1 antibody [CAL10] - Rat IgG2a (Chimeric) (AB279294)

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) (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

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

• WB

Supplier Data

Western blot - Anti-PD-L1 antibody [CAL10] - Rat IgG2a (Chimeric) (AB279294)

Blocking/Dilution buffer : 5% NFDM/TBST.

All lanes:

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

Lane 1:

CHO-S (Chinese hamster ovary epithelial cell) whole cell lysate at 20 µg

Lane 2:

CHO-PD-L1 (PD-L1 stably expressed Chinese hamster ovary epithelial cell) whole cell lysate at 20 µg

Lane 3:

Human placenta tissue lysate at 20 µg

Lane 4:

NCI-H1299 (human lung carcinoma epithelial cell), whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rat IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rat-igg-h-l-hrp-ab205720'>ab205720</a>) at 1/5000 dilution

Predicted band size: 33 kDa

Observed band size: 40-60 kDa

false

Exposure time: 8s

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-PD-L1 antibody [CAL10] - Rat IgG2a (Chimeric) (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 Cyt (Intra)

Supplier Data

Flow Cytometry (Intracellular) - Anti-PD-L1 antibody [CAL10] - Rat IgG2a (Chimeric) (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.