Anti-Mesothelin antibody [EPR19025-42]

7 product images

• 

  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 (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) (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.

• 

  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] (Anti-GAPDH antibody [6C5] - Loading Control 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 (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed (Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed 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

  Performed under reducing conditions.

  Predicted band size: 69 kDa

  Observed band size: 45 kDa

• 

  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) (VeriBlot for IP Detection Reagent (HRP) ab131366) at 1/4000 dilution

  Lane 2: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/100000 dilution

  Developed using the ECL technique.

  Predicted band size: 69 kDa

  Observed band size: 40 kDa, 69 kDa

  Exposure time: 30s

• 

  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) (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 (Rabbit IgG, monoclonal [EPR25A] - Isotype Control 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

• 

  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.

• 

  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 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.