Anti-Mesothelin antibody [EPR19025-42]

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Mesothelin antibody [EPR19025-42] (AB196235)

Immunohistochemical analysis of paraffin-embedded human ovarian adenocarcinoma tissue labeling Mesothelin with ab196235 at 1/1500 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) Ready to use. Cytoplasmic staining on tumor cells of human ovarian adenocarcinoma (PMID : 17945478). Counter stained with Hematoxylin.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) Ready to use.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• Flow Cyt

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Flow Cytometry - Anti-Mesothelin antibody [EPR19025-42] (AB196235)

Flow cytometric analysis of HeLa (human epithelial cell line from cervix adenocarcinoma) cell line labeling Mesothelin with ab196235 at 1/50 dilution (red) compared with a Rabbit IgG, monoclonal [EPR25A] - Isotype Control (ab172730) (black) and an unlabeled control (cells without incubation with primary antibody and secondary antibody) (blue). Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (ab150077) at 1/2000 dilution was used as the secondary antibody.

Total viable cells were gated for the image.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Mesothelin antibody [EPR19025-42] (AB196235)

Immunohistochemical analysis of paraffin-embedded human mesothelioma tissue labeling Mesothelin with ab196235 at 1/1500 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) Ready to use. Membranous and cytoplasmic staining on human mesothelioma (PMID : 17945478). Counter stained with Hematoxylin.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) Ready to use.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• Flow Cyt

Lab

Flow Cytometry - Anti-Mesothelin antibody [EPR19025-42] (AB196235)

Flow cytometry overlay histogram showing left HeLa positive cells and right negative PC3 stained with ab196235 (red line). The cells were incubated in 1x PBS containing 10% normal goat serum to block non-specific protein-protein interactionfollowed by the antibody (ab196235) (1x 106 in 100μl at 5.0 μg/ml (1/410)) for 30min on ice.

The secondary antibody Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed was incubated at 1/4000 for 30min on ice

Isotype control antibody (black line) was Recombinant Rabbit IgG, monoclonal [EPR25A] - Isotype Control used at the same concentration and conditions as the primary antibody. Unlabelled sample (blue line) was also used as a control.

Acquisition of >5000 events were collected using a 50 mW Blue laser (488nm) and 525/40 bandpass filter.

• IP

Supplier Data

Immunoprecipitation - Anti-Mesothelin antibody [EPR19025-42] (AB196235)

Mesothelin was immunoprecipitated from 0.35 mg of HeLa (human epithelial cell line from cervix adenocarcinoma) lysate with ab196235 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab196235 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/10000 dilution.

Lane 1 : HeLa whole cell lysate 10 μg (Input).

Lane 2 : ab196235 IP in HeLa whole cell lysate.

Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab196235 in HeLa whole cell lysate.

Exposure time : 3 minutes.

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

All lanes:

Immunoprecipitation - Anti-Mesothelin antibody [EPR19025-42] (ab196235)

Predicted band size: 69 kDa

Observed band size: 40 kDa,69 kDa

false

• WB

Supplier Data

Western blot - Anti-Mesothelin antibody [EPR19025-42] (AB196235)

Blocking/Dilution buffer : 5% NFDM/TBST.

The molecular mass is consistent with what has been described in the literature (PMID : 24146039).

All lanes:

Western blot - Anti-Mesothelin antibody [EPR19025-42] (ab196235) at 1/1000 dilution

Lane 1:

Human ovary cancer lysate at 20 µg

Lane 2:

HeLa (human epithelial cell line from cervix adenocarcinoma) cell lysate at 20 µg

Secondary

Lane 1:

Western blot - VeriBlot for IP Detection Reagent (HRP) (<a href='/en-us/products/reagents/veriblot-for-ip-detection-reagent-hrp-ab131366'>ab131366</a>) at 1/4000 dilution

Lane 2:

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: 69 kDa

Observed band size: 40 kDa,69 kDa

true

Exposure time: 30s

• WB

Lab

Western blot - Anti-Mesothelin antibody [EPR19025-42] (AB196235)

False colour image of Western blot : Anti-Mesothelin antibody [EPR19025-42] staining at 1/1000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab196235 was shown to bind specifically to Mesothelin. 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-Mesothelin antibody [EPR19025-42] (ab196235) at 1/1000 dilution

Lane 1:

OVCAR-3 cell lysate at 20 µg

Lane 2:

HeLa cell lysate at 20 µg

Lane 3:

A549 cell lysate at 20 µg

Lane 4:

PC-3 cell lysate at 20 µg

Predicted band size: 69 kDa

Observed band size: 45 kDa

false