All lanes : Anti-Tuberin antibody [Y320] (ab32554) at 1/5000 dilution (purified)

Lane 1 : HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate boiled
Lane 2 : HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate unboiled
Lane 3 : Mouse brain lysate boiled
Lane 4 : Mouse brain lysate unboiled
Lane 5 : Rat brain lysate boiled
Lane 6 : Rat brain lysate unboiled

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Predicted band size: 200 kDa
Observed band size: 200 kDa

The unboiled lysate are recommended to be used in SDS-PAGE, in order to  prevent membrane protein aggregation.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of rat liver tissue sections labeling Tuberin with purified ab32554 at 1/100 dilution (2.97 µg/mL). Heat mediated antigen retrieval using Bond™ Epitope Retrieval Solution 2 (pH 9.0). Rabbit specific IHC polymer detection kit HRP/DAB (ab209101) was used as the secondary antibody. Negative control: PBS instead of the primary antibody. Hematoxylin was used as a counterstain.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of mouse liver tissue sections labeling Tuberin with purified ab32554 at 1/100 dilution (2.97 µg/mL). Heat mediated antigen retrieval using Bond™ Epitope Retrieval Solution 2 (pH 9.0). Rabbit specific IHC polymer detection kit HRP/DAB (ab209101) was used as the secondary antibody. Negative control: PBS instead of the primary antibody. Hematoxylin was used as a counterstain.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human lung carcinoma tissue sections labeling Tuberin with purified ab32554 at 1/100 dilution (2.97 µg/mL). Heat mediated antigen retrieval using Bond™ Epitope Retrieval Solution 2 (pH 9.0). Rabbit specific IHC polymer detection kit HRP/DAB (ab209101) was used as the secondary antibody. Negative control: PBS instead of the primary antibody. Hematoxylin was used as a counterstain.

Lane 1: Wild-type HAP1 whole cell lysate (20 µg)              
Lane 2: Tuberin knockout HAP1 whole cell lysate (20 µg)
Lane 3: HeLa whole cell lysate (20 µg)

Lanes 1 - 4: Merged signal (red and green). Green -  unpurified ab32554 observed at 180 kDa. Red - loading control, ab18058, observed at 130 kDa.

ab32554 was shown to specifically react with tuberin in wild-type HAP1 cells. No band was observed when tuberin knockout samples were used. Wild-type and tuberin knockout samples were subjected to SDS-PAGE.  Ab32554 and ab18058 (Mouse anti Vinculin loading control) were incubated overnight at 4°C at 1/1000 dilution and 1/10000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed ab216776 secondary antibodies at 1/10000 dilution for 1 hour at room temperature before imaging.

Flow Cytometry analysis of Jurkat (Human T cell leukemia T lymphocyte) cells labeling Tuberin with purified ab32554 at 1/30 dilution (10 µg/mL) (Red). Cells were fixed with 4% Paraformaldehyde and permeabilised with 90% Methanol. A Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) secondary antibody was used at 1/2000. Isotype control - Rabbit monoclonal IgG (Black). Unlabeled control - Cell without incubation with primary antibody and secondary antibody (Blue).