All lanes : Anti-G3BP antibody [EPR13986(B)] (ab181150) at 1/1000 dilution

Lane 1 : Wild-type A431 whole cell lysate
Lane 2 : G3BP1 knockout A431 whole cell lysate
Lane 3 : Jurkat whole cell lysate
Lane 4 : HEK-293 whole cell lysate

Lysates/proteins at 20 µg per lane.

Predicted band size: 52 kDa

Lanes 1 - 4: Merged signal (red and green). Green - ab181150 observed at 68 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab181150 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. Ab181150 and ab8245 (Mouse anti-GAPDH loading control) were incubated overnight at 4°C at 1/1000 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.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of Human hepatocellular cancer tissue sections labeling G3BP with purified ab181150 at 1/50 dilution (6.86 µg/ml). Heat mediated antigen retrieval was performed using Citrate buffer, pH 6.0. ImmunoHistoProbe one step HRP Polymer (ready to use) was used as the secondary antibody. Negative control: PBS instead of the primary antibody. Hematoxylin was used as a counterstain.

All lanes : Anti-G3BP antibody [EPR13986(B)] (ab181150) at 1/10000 dilution (Purified)

Lane 1 : HEK-293 (Human embryonic kidney epithelial cell) whole cell lysates
Lane 2 : Ramos (Human Burkitt's lymphoma B lymphocyte) whole cell lysates
Lane 3 : PC-12 (Rat adrenal gland pheochromocytoma) whole cell lysates
Lane 4 : NIH/3T3 (Mouse embryonic fibroblast) whole cell lysates

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: 52 kDa
Observed band size: 68 kDa why is the actual band size different from the predicted?

Immunocytochemistry/ Immunofluorescence analysis of HeLa (Human cervix adenocarcinoma epithelial cell) cells labeling G3BP with purified ab181150 at 1/500 dilution (0.7 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] - Microtubule Marker (Alexa Fluor^® 594) at 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.

All lanes : Anti-G3BP antibody [EPR13986(B)] (ab181150) at 1/5000 dilution (unpurified)

Lane 1 : Jurkat cell lysate with NFDM/TBST
Lane 2 : Ramos cell lysate with NFDM/TBST

Lysates/proteins at 20 µg per lane.

Blocking peptides at 5 % per lane.

Secondary
All lanes : Peroxidase conjugated Goat anti-Rabbit IgG (H+L) at 1/1000 dilution

Predicted band size: 52 kDa
Observed band size: 68 kDa why is the actual band size different from the predicted?

ab181150 (purified) at 1/20 dilution (2ug) immunoprecipitating G3BP in Ramos whole cell lysate. Ramos (Human Burkitt's lymphoma B lymphocyte) whole cell lysate 10ug
Lane 2 (+): ab181150 & Ramos whole cell lysate
Lane 3 (-): Rabbit monoclonal IgG (ab172730) instead of ab181150 in Ramos whole cell lysate
For western blotting, VeriBlot for IP secondary antibody (HRP) (ab131366) was used at 1/1000 dilution.
Blocking and diluting buffer: 5% NFDM/TBST.

Flow Cytometry analysis of HeLa (Human cervix adenocarcinoma epithelial cell) cells labeling G3BP with purified ab181150 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).

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of Rat kidney tissue sections labeling G3BP with purified ab181150 at 1/50 dilution (6.86 µg/ml). Heat mediated antigen retrieval was performed using Citrate buffer, pH 6.0. ImmunoHistoProbe one step HRP Polymer (ready to use) 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 kidney tissue sections labeling G3BP with purified ab181150 at 1/50 dilution (6.86 µg/ml). Heat mediated antigen retrieval was performed using Citrate buffer, pH 6.0. ImmunoHistoProbe one step HRP Polymer (ready to use) was used as the secondary antibody. Negative control: PBS instead of the primary antibody. Hematoxylin was used as a counterstain.

Immunofluorescent analysis of 4% paraformaldehyde fixed 293 cells staining G3BP using ab181150 (unpurified) at 1/100 dilution, and Alexa Fluor®555 stained Goat anti Rabbit IgG at 1/200 dilution as a secondary antibody (red). Dapi counterstain (blue)

All lanes : Anti-G3BP antibody [EPR13986(B)] (ab181150) at 1/1000 dilution (unpurified)

Lane 1 : Hela cell lysate with NFDM/TBST
Lane 2 : 293 cell lysate with NFDM/TBST

Lysates/proteins at 20 µg per lane.

Blocking peptides at 5 % per lane.

Secondary
All lanes : Peroxidase conjugated Goat anti-Rabbit IgG (H+L) at 1/1000 dilution

Predicted band size: 52 kDa
Observed band size: 68 kDa why is the actual band size different from the predicted?

Immunohistochemical analysis of paraffin-embedded Human colon tissue staining G3BP using ab181150 (unpurified) at 1/100 dilution, and prediluted HRP Polymer for Rabbit IgG as a secondary antibody with Hematoxylin counterstain.

Western blot analysis of G3BP in immunoprecipitation pellets from Ramos cell lysate, using ab181151 at a 1/50 dilution. Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated was used as a secondary antibody at 1/1000 dilution. Blocking/dilution buffer and concentration: 5% NFDM/TBST

Anti-G3BP antibody [EPR13986(B)] (ab181150) at 1/50 dilution (unpurified) + Immunoprecipitate from Ramos cell lysate using ab181150 with NFDM/TBST at 5 %

Secondary
Peroxidase conjugated Goat anti-Rabbit IgG (H+L) at 1/1000 dilution