Recombinant Anti-PTEN antibody [EPR9941-2] (ab170941)

Blocking and diluting buffer: 5% NFDM/TBST. 

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of Rat cerebrum tissue sections labeling PTEN with Purified ab170941 at 1:50 dilution (5.3 µg/ml). Heat mediated antigen retrieval was performed using ab93684 (Tris/EDTA buffer, pH 9.0)ImmunoHistoProbe one step HRP Polymer (ready to use)was used as the secondary antibody.Negative control:PBS instead of the primary antibody.Hematoxylinwas used as a counterstain

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of Mouse cerebrum tissue sections labeling PTEN with Purified ab170941 at 1:50 dilution (5.3 µg/ml). Heat mediated antigen retrieval was performed using ab93684 (Tris/EDTA buffer, pH 9.0)ImmunoHistoProbe one step HRP Polymer (ready to use)was used as the secondary antibody.Negative control:PBS instead of the primary antibody.Hematoxylinwas used as a counterstain

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of Human pancreas tissue sections labeling PTEN with Purified ab170941 at 1:50 dilution (5.3 µg/ml). Heat mediated antigen retrieval was performed using ab93684 (Tris/EDTA buffer, pH 9.0)ImmunoHistoProbe one step HRP Polymer (ready to use)was used as the secondary antibody.Negative control:PBS instead of the primary antibody.Hematoxylinwas used as a counterstain.

Lane 1: Wild-type HAP1 cell lysate (20 µg)
Lane 2: PTEN knockout HAP1 cell lysate (20 µg)
Lanes 1 and 2: Merged signal (red and green). Green - ab170941 observed at 47 kDa. Red - loading control, ab8245, observed at 37 kDa.

Unpurified ab170941 was shown to specifically react with PTEN in wild-type HAP1 cells. No band was observed when PTEN knockout samples were used. Wild-type and PTEN knockout samples were subjected to SDS-PAGE, ab170941 and ab8245 (loading control to GAPDH) were diluted 1/500 and 1/1000 respectively and incubated overnight at 4°C. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (ab216776) secondary antibodies at 1/10,000 dilution for 1hr at room temperature before imaging.

Immunohistochemical analysis of paraffin-embedded Human breast carcinoma tissue labeling PTEN with unpurifed ab170941 at 1/50 dilution. Heat mediated antigen retrieval was performed using citrate buffer pH 6 before commencing with IHC staining protocol.

Intracellular Flow Cytometry analysis of HeLa (human cervix adenocarcinoma) cells labeling PTEN with purified ab170941 at 1/120 dilution (10ug/ml) (red). Cells were fixed with 80% methanol and permeabilised with 0.1% Tween-20. A Goat anti rabbit IgG (Alexa Fluor^® 488) (ab150077) (1/2000 dilution) was used as the secondary antibody. Rabbit monoclonal IgG (Black) (ab172730) was used as the isotype control, cells without incubation with primary antibody and secondary antibody (Blue) were used as the unlabeled control. 

Immunohistochemical analysis of paraffin-embedded Human colon tissue labeling PTEN with unpurifed ab170941 at 1/50 dilution. Heat mediated antigen retrieval was performed using citrate buffer pH 6 before commencing with IHC staining protocol.