Recombinant Anti-PD-L1 antibody [CAL10] - Mouse IgG2a (Chimeric) (ab279293)

All lanes : 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
Lane 2 : Wild-type A549 Vehicle Control IFN-gamma (0 ng/mL, 48 h) cell lysate
Lane 3 : CD274 knockout A549 Treated IFN-gamma (100 ng/mL, 48 h) cell lysate
Lane 4 : CD274 knockout A549 Vehicle Control IFN-gamma (0 ng/mL, 48 h) cell lysate
Lane 5 : Human Placenta cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Observed band size: 45-65 kDa why is the actual band size different from the predicted?

False colour image of Western blot: Anti-PD-L1 antibody [CAL10] – Mouse IgG2a (Chimeric) 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.

All lanes : Anti-PD-L1 antibody [CAL10] - Mouse IgG2a (Chimeric) (ab279293) at 1/1000 dilution

Lane 1 : CHO-S (Chinese hamster ovary epithelial cell) whole cell lysate
Lane 2 : CHO-PD-L1 (PD-L1 stably expressed Chinese hamster ovary epithelial cell) whole cell lysate
Lane 3 : Human placenta tissue lysate
Lane 4 : NCI-H1299 (human lung carcinoma epithelial cell), whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Peroxidase-Conjugated Goat anti-Mouse IgG (H+L) at 1/5000 dilution

Observed band size: 40-60 kDa why is the actual band size different from the predicted?

Blocking/Dilution buffer: 5% NFDM/TBST.

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 ab279293, 1ug/ml, for 15 mins at room temperature. A rabbit anti-mouse IgG2a, 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

Immunocytochemical analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100-fixed permeabilized CHO-PD-L1 cells labeling PD-L1 with ab279293 at 1/100 dilution, followed by ab150113 Goat Anti-Mouse IgG H&L (Alexa Fluor^® 488) antibody 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: ab279293 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 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 ab279293 at 1/50 dilution (0.1µg).

Goat Anti-Mouse IgG (Alexa Fluor^® 488, ab150113) at 1/2000 dilution was used as the secondary antibody.