The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
1/10000 - 1/50000. Detects a band of approximately 68 kDa (predicted molecular weight: 52 kDa).
1/50 - 1/100.
1/250 - 1/500.
1/10. ab172730-Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody.
1/20 - 1/40.
May be a regulated effector of stress granule assembly. Phosphorylation-dependent sequence-specific endoribonuclease in vitro. Cleaves exclusively between cytosine and adenine and cleaves MYC mRNA preferentially at the 3'-UTR. ATP- and magnesium-dependent helicase. Unwinds preferentially partial DNA and RNA duplexes having a 17 bp annealed portion and either a hanging 3' tail or hanging tails at both 5'- and 3'-ends. Unwinds DNA/DNA, RNA/DNA, and RNA/RNA substrates with comparable efficiency. Acts unidirectionally by moving in the 5' to 3' direction along the bound single-stranded DNA.
Phosphorylated exclusively on serine residues. Hyperphosphorylated in quiescent fibroblasts. Hypophosphorylation leads to a decrease in endoribonuclease activity (By similarity). RASA1-dependent phosphorylation of Ser-149 induces a conformational change that prevents self-association. Dephosphorylation after HRAS activation is required for stress granule assembly. Ser-149 phosphorylation induces partial nuclear localization. Arg-435 is dimethylated, probably to asymmetric dimethylarginine.
Cytoplasm. Cytoplasm > cytosol. Cell membrane. Nucleus. Cytoplasmic in proliferating cells, can be recruited to the plasma membrane in exponentially growing cells (By similarity). Cytosolic and partially nuclear in resting cells. Recruited to stress granules (SGs) upon either arsenite or high temperature treatment. Recruitment to SGs is influenced by HRAS.
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.
Western blot - Anti-G3BP antibody (ab181149)
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 Peroxidase conjugated Goat anti-Rabbit IgG (H+L) at 1/1000 dilution
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.
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 Cytometry - Anti-G3BP antibody (ab181149)
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