HTRF Human Phospho-Y174 VAV1 assay principle

The Phospho-Y174 VAV1 assay measures VAV1 when phosphorylated at Tyr174. Unlike Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis, or transfer. The assay uses 2 antibodies, one labeled with a donor fluorophore and the other with an acceptor. The first antibody was selected for its specific binding to the phosphorylated motif on the protein, the second for its ability to recognize the protein independently of its phosphorylation state. Protein phosphorylation enables an immune-complex formation involving both labeled antibodies, and which brings the donor fluorophore into close proximity to the acceptor, thereby generating a FRET signal. Its intensity is directly proportional to the concentration of phosphorylated protein present in the sample, and provides a means of assessing the protein's phosphorylation state under a no-wash assay format.

HTRF Human Phospho-Y174 VAV1 one-plate assay protocol

Detection of Phospho-Y174 VAV1 with HTRF reagents can be performed in a single plate used for culturing, stimulation, and lysis. No washing steps are required. This HTS designed protocol enables miniaturization while maintaining robust HTRF quality.

HTRF Human Phospho-Y174 VAV1 two-plate assay protocol

The two-plate protocol involves culturing cells in a 96-well plate before lysis, then transferring lysates into a low volume detection plate (either HTRF 384-lv or 96-lv plate) before the addition of HTRF Phospho-Y174 VAV1 detection reagents. This protocol enables the cells' viability and confluence to be monitored.

Dose-response of the HTRF Phospho/Total VAV1 assay on cells treated with pervanadate

Jurkat cells (100,00 cells/well) were plated at 25 µL in a 96-well plate in complete culture medium and treated with 5 µL of increasing concentrations of pervanadate diluted in media, and incubated for 30 min at 37ºC, 5% CO2.

The cells were lysed with 10 µL of 4x lysis buffer #1 for 45 minutes at RT.  After lysis, 16 µL of lysate were transferred into a low volume white microplate before the addition of 2 µL each of the donor and acceptor HTRF PVAV1 or Total VAV1 detection reagents.  The HTRF signal was recorded after an overnight incubation.

VAV1 signaling pathway

Conformational changes on the intracellular portion of the receptor promotes the recruitment of tyrosine kinases Lck and ZAP-70, which phosphorylate its tyrosine domains as well as other neighboring signaling proteins, like LAT (Linker for Activation of T cells).

Phosphorylated LAT then recruits VAV1 and promotes its phosphorylation and activation. Phospho-VAV1 goes on to catalyze GDP/GTP exchanges on proteins of the Rho family, especially Rac1 and Cdc42. These Rho proteins then trigger a cascade of downstream signaling events which include sending transcription factors to the nucleus to promote cytoskeletal reorganization, cell adhesion, migration, and proliferation, as well as other immune-cell mechanisms promoting the elimination of pathogens or compromised cells.