Lysates prepared from COS cells overexpressing human wild-type PAG3 and cotransfected with mouse Pyk2 were immunoblotted in the presence of non-phosphopeptide corresponding to the immunogen, a generic phosphotyrosine-containing peptide or the phosphopeptide immunogen. The data show that only the peptide corresponding to phospho Y724 PAG3 blocks the signal, and the signal is absent when mutant PAG3 extracts are used, demonstrating the specificity of the antibody.
This antibody was purified from rabbit serum by sequential epitope specific chromatography. The antibody has been negatively preadsorbed using a non phosphopeptide corresponding to the site of phosphorylation to remove antibody that is reactive with non phosphorylated PAG3 protein. The final product is generated by affinity chromatography using a PAG3 derived peptide that is phosphorylated at tyrosine 724.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
1/1000. Detects a band of approximately 160 kDa (predicted molecular weight: 112 kDa).
Activates the small GTPases ARF1, ARF5 and ARF6. Regulates the formation of post-Golgi vesicles and modulates constitutive secretion. Modulates phagocytosis mediated by Fc gamma receptor and ARF6. Modulates PXN recruitment to focal contacts and cell migration.
Detected in heart, brain, placenta, kidney, monocytes and pancreas.
ANK repeat and PH domain-containing protein 2 antibody
Arf-GAP with SH3 domain antibody
ArfGAP with SH3 domain, ankyrin repeat and PH domain 2 antibody
centaurin, beta 3 antibody
DDEF 2 antibody
Development and differentiation enhancing factor 2 antibody
Development and differentiation-enhancing factor 2 antibody
PAG 3 antibody
Pap alpha antibody
Paxillin associated protein with ARFGAP activity 3 antibody
Paxillin-associated protein with ARF GAP activity 3 antibody
Pyk2 C terminus associated protein antibody
Pyk2 C-terminus-associated protein antibody
SHAG 1 antibody
Western blot - PAG3 (phospho Y724) antibody (ab18031)
Predicted band size : 112 kDa
Western blot using Ab18031 on COS cell lysate overexpressing human PAG3.
Lane 1: wild-type PAG3 alone
Lane 2: wild-type PAG3 cotransfected with mouse PyK2
Lane 3: wild-type PAG3 cotransfected with mouse PyK2. Antibody blocked with the non phosphopeptde corresponding to the immunogen
Lane 4: wild-type PAG3 cotransfected with mouse PyK2. Antibody blocked with the generic phosphotyrosine corresponding to the immunogen
Lane 5: wild-type PAG3 cotransfected with mouse PyK2. Antibody blocked with the generic phosphopeptide immunogen
Lane 6: mutant [Y724/Y763] PAG3 cotransfected with Pyk2
10-30 micrograms of cell lysate can be loaded when using similar lysates with this antibody. Samples were run using SDS-PAGE on a 8% polyacrylamide gel and transferred to PVDF. Membranes were blocked with a 5% BSA-TBST buffer for one hour at room temperature, then incubated with PAG3 antibody Ab18031 for two hours at room temperature in a 3% BSA-TBST buffer, following prior incubation with blocking peptides. After washing, membranes were incubated with goat F(ab’)2 anti-rabbit IgG HRP conjugate in 3% BSA-TBST buffer, and bands were detected using the Pierce SuperSignal method.
Only the phospho peptide immunogen blocks the antibody, confirming its specificity for the phospho epitope tyrosine 724.