HTRF STAT5 P-Y694 KIT - 500 PTS

HTRF compared to western blot using phospho-STAT5 (Tyr694) assay kit

TF1 cells were grown in a T175 flask at 37°C until concentration reached 1.5M/ml of cell culture medium, then stimulated with IL3 for 10min. Cells were incubated for 30 minutess for lysis. Serial dilutions (1:2) were analyzed in parallel by HTRF and Western-blot. Note that 128KC corresponds roughly to 32µg of proteins. By using HTRF phospho-STAT5 (Tyr694) only 2000 cells are sufficient for minimal signal detection while 8000 cells are needed for a Western blot signal. The HTRF assay is at least 4-fold more sensitive than the Western blot and shows optimal correlation.

 

HTRF assay compared to ELISA using phospho-STAT5 (Tyr694) assay kit

TF1 cells were cultured and treated with increasing concentrations of a JAK inhibitor and stimulated with IL3 for 10min. Following lysis, serial dilutions of collected supernatant were analyzed in parallel by HTRF and ELISA. The results displayed in the graph show that for measuring STAT5 phosphorylation HTRF is more sensitive than ELISA, offers a bigger assay window and is more reproducible. Despite a good correlation between ELISA and HTRF, the overall performance of HTRF is more favorable.

 

Pharmacological response in TF1 cells using a two plate assay protocol

TF1 cells were plated in 25µl of cell culture medium treated for 30min with 5µl of increasing concentrations of the JAK inhibitor Lestaurtinib. After removal of cell culture medium, 10µl of 4X supplemented lysis buffer were added and incubated for 30 minutes prior to the addition of the HTRF detection reagents. 16µl of lysates were transferred in a 384sv white plate for analysis of STAT5 phosphorylation. The results clearly demonstrate, that the high sensitivity of the assay allows a 4-fold lower cell quantity to be used without a significant decrease of assay window.

 

Pharmacological response in TF1 cells using a two plate assay protocol

TF1 cells were plated in 25µl of cell culture medium treated for 30min with 5µl of increasing concentrations of the JAK inhibitor Lestaurtinib. After removal of cell culture medium, 10µl of 4X supplemented lysis buffer were added and incubated for 30 minutes prior to the addition of the HTRF detection reagents. 16µl of lysates were transferred in a 384sv white plate for analysis of STAT5 phosphorylation. The results clearly demonstrate, that the high sensitivity of the assay allows a 4-fold lower cell quantity to be used without a significant decrease of assay window.

 

Pharmacological response in HeLa cells using a two plate assay protocol

100KC HeLa cells were plated in 25µl of cell culture medium treated for 2h with 5µl of increasing concentrations of the JAK inhibitor Lestaurtinib and then stimulated with 20 nM IFN for 10min. After removal of cell culture medium, 10µl of 4X supplemented lysis buffer were added and incubated for 30 minutes prior to the addition of the HTRF detection reagents. 16µl of lysates were transferred in a 384sv white plate for analysis of STAT5 phosphorylation.