Anti-PD-L1 antibody [RM1012]

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PD-L1 antibody [RM1012] (AB282458)

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

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PD-L1 antibody [RM1012] (AB282458)

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

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PD-L1 antibody [RM1012] (AB282458)

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

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PD-L1 antibody [RM1012] (AB282458)

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

• IP

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Immunoprecipitation - Anti-PD-L1 antibody [RM1012] (AB282458)

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

All lanes:

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

Predicted band size: 33 kDa

Observed band size: 40-60 kDa

false

• WB

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Western blot - 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.

All lanes:

Western blot - Anti-PD-L1 antibody [RM1012] (ab282458) at 1/10000 dilution

Lane 1:

Untreated CD274 (PD-L1) KO A549 (Human lung carcinoma epithelial cell) cell lysate at 20 µg

Lane 2:

CD274 (PD-L1) KO A549 treated with 100 ng/ml human IFN gamma for 48 hours cell lysate at 20 µg

Lane 3:

Untreated wild-type A549 cell lysate at 20 µg

Lane 4:

Wild-type A549 treated with 100ng/ml human IFN gamma for 48 hours cell lysate at 20 µg

Lane 5:

MDA-MB-231 (Human breast adenocarcinoma epithelial cell) cell lysate at 20 µg

Lane 6:

NCI-H1975 (Human adenocarcinoma lung epithelial cell) cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-irdye-800cw-preadsorbed-ab216773'>ab216773</a>) at 1/100000 dilution

Predicted band size: 33 kDa

Observed band size: 40-60 kDa

false

Exposure time: 3min

• WB

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Western blot - Anti-PD-L1 antibody [RM1012] (AB282458)

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

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

All lanes:

Western blot - Anti-PD-L1 antibody [RM1012] (ab282458) at 1/1000 dilution

Lane 1:

NCI-H1975 (Human adenocarcinoma lung epithelial cell) whole cell lysate at 20 µg

Lane 2:

MDA-MB-231 (Human breast adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Lane 3:

MCF7 (Human breast adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Lane 4:

Human placenta lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Predicted band size: 33 kDa

Observed band size: 40-60 kDa

false

• Flow Cyt

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Flow Cytometry - Anti-PD-L1 antibody [RM1012] (AB282458)

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.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-PD-L1 antibody [RM1012] (AB282458)

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.

• I-ELISA

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Indirect ELISA - Anti-PD-L1 antibody [RM1012] (AB282458)

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.