Recombinant Anti-PD-L1 antibody [RM1012] (ab282458)

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

False colour image of Western blot: Anti-PD-L1 antibody [RM1012] (ab282458) staining at 1/10, 000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in red.

In Western blot, ab282458 was shown to bind specifically to PD-L1. Target band was observed at 40-60 kDa in wild-type A549 treated with IFN gamma cell lysates with no signal observed at this size in PD-L1 knockout cell line ab267054 (knockout cell lysate ab256831). To generate this image, wild-type and CD274 (PD-L1) knockout A549 cell lysates were analyzed. 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 (IRDye^® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (ab216776) at 1/20000 dilution.

Immunohistochemical analysis of paraffin-embedded Human lung cancer tissue labelling PD-L1 with ab282458 at 1/500 (0.876 ug/ml) dilution followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Positive staining on human lung cancer. The section was incubated with ab282458 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin.

Secondary antibody only control: Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized CHO-PDL1 cells labelling PD-L1 with ab282458 at 1/1000 (0.438 ug/ml) dilution, followed by ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) antibody at 1/1000  dilution (Green). Confocal image showing membranous staining in CHO-PDL1 cells and no staining in CHO-S cells.

ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 2.5ug/ml dilution (Red). The Nuclear counterstain was DAPI (Blue).

Secondary antibody only control: Secondary antibody is ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) at 1/1000  dilution.

Blocking and Diluting buffer and concentration: 5% NFDM/TBST.

Low expression control: MCF7 (PMID: 28184013, 31741201).

Flow cytometric analysis of CHO-S (Chinese hamster ovary epithelial cell (Blue)) / CHO-PDL1 (PD-L1 stably expessed Chinese hamster ovary epithelial cell) (Red)) cells labelling PD-L1 with ab282458 at 1/50 dilution (1ug). Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) at 1/2000 dilution was used as the secondary antibody. Gated on viable cells.

PD-L1 was immunoprecipitated from 0.35 mg NCI-H1975 (Human adenocarcinoma lung epithelial cell) whole cell lysate with ab282458 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab282458 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution.

Lane 1: NCI-H1975 (Human adenocarcinoma lung epithelial cell) whole cell lysate 10 ug

Lane 2: ab282458 IP in NCI-H1975 whole cell lysate

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab282458 in NCI-H1975 whole cell lysate

Blocking and dilution buffer and concentration: 5% NFDM/TBST.

Exposure time: 32 seconds

Immunohistochemical analysis of paraffin-embedded Human breast cancer tissue labelling PD-L1 with ab282458 at 1/500 (0.876 ug/ml) dilution followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Positive staining on human breast cancer. The section was incubated with ab282458 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin.

Secondary antibody only control: Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins

ELISA using ab282458 at varying antibody concentrations and antigen concentration at 1000 ng/ml. An Alkaline Phosphatase-conjugated AffiniPure Goat Anti-Rabbit IgG (H+L) (1/2500) was used as the secondary antibody.

Immunohistochemical analysis of paraffin-embedded Human placenta tissue labelling PD-L1 with ab282458 at 1/500 (0.876 ug/ml) dilution followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Positive staining on human placenta. The section was incubated with ab282458 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin.

Secondary antibody only control: Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins

Immunohistochemical analysis of paraffin-embedded Human tonsil tissue labelling PD-L1 with ab282458 at 1/500 (0.876 ug/ml) dilution followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Positive staining on human tonsil. The section was incubated with ab282458 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin.

Secondary antibody only control: Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins