重组Anti-Mesothelin抗体[EPR19025-42] - BSA and Azide free

• Flow Cyt

Supplier Data

Flow Cytometry - Anti-Mesothelin antibody [EPR19025-42] - BSA and Azide free (AB227810)

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab196235).

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Mesothelin antibody [EPR19025-42] - BSA and Azide free (AB227810)

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab196235).

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

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Mesothelin antibody [EPR19025-42] - BSA and Azide free (AB227810)

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab196235).

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] - BSA and Azide free (AB227810)

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (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] - BSA and Azide free (AB227810)

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab196235).

All lanes:

Immunoprecipitation - Anti-Mesothelin antibody [EPR19025-42] (<a href='/products/primary-antibodies/mesothelin-antibody-epr19025-42-ab196235'>ab196235</a>)

Predicted band size: 69 kDa

Observed band size: 40 kDa,69 kDa

false

• WB

Lab

Western blot - Anti-Mesothelin antibody [EPR19025-42] - BSA and Azide free (AB227810)

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (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] (<a href='/products/primary-antibodies/mesothelin-antibody-epr19025-42-ab196235'>ab196235</a>) 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