All lanes : Anti-G3BP antibody [EPR13985(B)] (ab181149) at 1/10000 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 - 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.

Immunocytochemistry/Immunofluorescence analysis of Jurkat (Human T cell leukemia cell line from peripheral blood) labeling G3BP with Purified ab181149 at 1/500 dilution (5 µg/ml). Cells were fixed with 4% PFA and permeabilized with 0.1% tritonX-100. ab150077 Goat anti rabbit IgG(Alexa Fluor^® 488) at 1/1000 dilution was used as the secondary antibody. Nuclei were counterstained with DAPI. PBS was used instead of the primary antibody as the negative control.

Immunohistochemical analysis of paraffin-embedded Human colon tissue staining G3BP using ab181149 at 1/100 dilution, and prediluted HRP Polymer for Rabbit IgG as a secondary antibody with Hematoxylin counterstain.

Perform heat mediated antigen retrieval with EDTA buffer pH 9 before commencing with IHC staining protocol.

All lanes : Anti-G3BP antibody [EPR13985(B)] (ab181149) at 1/20000 dilution

Lane 1 : Hela cell lysate with NFDM/TBST
Lane 2 : Jurkat cell lysate with NFDM/TBST
Lane 3 : Ramos cell lysate with NFDM/TBST
Lane 4 : 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 muscle tissue staining G3BP using ab181149 at 1/100 dilution, and prediluted HRP Polymer for Rabbit IgG as a secondary antibody with Hematoxylin counterstain.

Perform heat mediated antigen retrieval with EDTA buffer pH 9 before commencing with IHC staining protocol.

Immunofluorescence analysis of 4% paraformaldehyde fixed 293 cells staining G3BP using ab181149 at 1/500 dilution (red), and Goat anti rabbit IgG (Alexa Fluor® 555) at 1/200 dilution as a secondary antibody. Dapi was used for counterstaining (blue).

Flow cytometric analysis of 2% paraformaldehyde fixed Jurkat cells staining G3BP using ab181149 at 1/10 dilution, and FITC conjugated Goat anti rabbit IgG at 1/150 dilution as a secondary antibody (red curve). Rabbit monoclonal IgG was the negative control (green curve).

 

Western blot analysis of G3BP in immunoprecipitation pellets from Ramos cell lysate, using ab181149 at a 1/50 dilution. Anti-Rabbit IgG (HRP) specific to the non-reduced form of IgG was used as a secondary antibody at 1/1000 dilution. Blocking/dilution buffer and concentration: 5% NFDM/TBST