Recombinant Anti-G3BP antibody [EPR13985(B)] (ab181149)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human breast carcinoma tissue sections labeling G3BP with Purified ab181149 at 1:300 (0.4 µg/ml). Heat mediated antigen retrieval was performed using Heat mediated antigen retrieval using Bond™ Epitope Retrieval Solution 2 (pH 9.0) . Tissue was counterstained with Hematoxylin. Rabbit specific IHC polymer detection kit HRP/DAB (ab209101) secondary antibody was used at 1:0 dilution. PBS instead of the primary antibody was used as the negative control. 

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human breast tissue sections labeling G3BP with Purified ab181149 at 1:300 (0.4 µg/ml). Heat mediated antigen retrieval was performed using Heat mediated antigen retrieval using Bond™ Epitope Retrieval Solution 2 (pH 9.0) . Tissue was counterstained with Hematoxylin. Rabbit specific IHC polymer detection kit HRP/DAB (ab209101) secondary antibody was used at 1:0 dilution. PBS instead of the primary antibody was used as the negative control. 

Purified ab181149 at 1:20 dilution (0.6µg) immunoprecipitating G3BP in Ramos whole cell lysate.
Lane 1 (input): Ramos (Human Burkitt's lymphoma B lymphocyte) whole cell lysate 10 µg.
Lane 2 (+): ab181149 + Ramos whole cell lysate.
Lane 3 (-): Rabbit monoclonal IgG (ab172730) instead of ab181149 in Ramos whole cell lysate.
VeriBlot for IP Detection Reagent (HRP)(ab131366) (1:1000 dilution) was used for Western blotting.
Blocking Buffer and concentration: 5% NFDM/TBST.
Diluting buffer and concentration: 5% NFDM/TBST.
Observed band size: kDa

Flow Cytometry analysis of Jurkat (Human T cell leukemia T lymphocyte) cells labelling G3BP with Purified ab181149 at 1:20 dilution (5 µg/ml) (Red). Cells were fixed with 4% Paraformaldehyde and permeabilised with 90% Methanol. A Goat anti rabbit IgG (Alexa Fluor® 488, ab150081) secondary antibody was used at 1:2000. Isotype control - Rabbit monoclonal IgG (Black). Unlabelled control - Cell without incubation with primary antibody and secondary antibody (Blue).

Immunocytochemistry analysis of Jurkat (Human T cell leukemia T lymphocyte) cells labeling G3BP with Purified ab181149 at 1:50 dilution (2.4 µg/ml). Cells were fixed in 4% Paraformaldehyde and permeabilized with 0.1% tritonX-100. Cells were counterstained with Ab195889 Anti-alpha Tubulin antibody [DM1A]+H21:L21 - Microtubule Marker (Alexa Fluor® 594) 1:200 (2.5 µg/ml). Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) was used as the secondary antibody at 1:1000 (2 µg/ml) dilution. DAPI (blue) was used as nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.

Lanes 1 - 4: Merged signal (red and green). Green - ab181149 observed at 68 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab181149 was shown to specifically react with G3BP1 in wild-type A431 cells as signal was lost in G3BP1 knockout cells. Wild-type and G3BP1 knockout samples were subjected to SDS-PAGE. The membrane was blocked with 3% Milk. Ab181149 and ab8245 (Mouse anti-GAPDH loading control) were incubated overnight at 4°C at 1/10000 dilution and 1/20000 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 1/20000 dilution for 1 hour at room temperature before imaging.