Figure 1. Treatment of Jurkat cells with anti-CD3 stimulates phosphorylation of LAT at Tyr191 as detected by the PathScan® Phospho-LAT (Tyr220) Sandwich ELISA Kit #7936, but does not affect the level of total LAT. Jurkat cells were starved for 48 hours and treated with 10 µg/ml anti-CD3 for 2 minutes at 37ºC. The absorbance readings at 450 nm are shown in the top figure, while the western blots, using LAT Antibody #9166 (left panel) or Phospho-LAT (Tyr220) Antibody #3584 (right panel), are shown in the bottom figure.
Figure 2. The relationship between lysate protein concentration from untreated and anti-CD3-treated Jurkat cells and the absorbance at 450 nm is shown.
|7936C||1 Kit (96 assays)||$ 513|
|Product Includes||Volume||Solution Color|
|LAT Mouse mAb Coated Microwells||96 tests|
|Phospho-LAT (Tyr220) Rabbit Detection Antibody||1 ea||Green (Lyophilized)|
|Anti-rabbit IgG, HRP-linked Antibody (ELISA Formulated)||1 ea||Red (Lyophilized)|
|Detection Antibody Diluent||11 ml||Green|
|HRP Diluent||11 ml||Red|
|TMB Substrate 7004||11 ml|
|STOP Solution 7002||11 ml|
|Sealing Tape||2 ea|
|ELISA Wash Buffer (20X) 9801||25 ml|
|ELISA Sample Diluent||25 ml||Blue|
|Cell Lysis Buffer (10X) 9803||15 ml|
The PathScan® Phospho-LAT (Tyr220) Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of phospho-LAT (Tyr220). A LAT mouse antibody has been coated onto the microwells. After incubation with cell lysates, LAT protein (phosphorylated and non-phosphorylated) is captured by the coated antibody. Following extensive washing, a phospho-LAT (Tyr220) rabbit detection antibody is added to detect the captured phospho-LAT (Tyr220). HRP-linked anti-rabbit antibody is then used to recognize the bound detection antibody. The HRP substrate TMB is added to develop color. The magnitude of the absorbance for this developed color is proportional to the quantity of phospho-LAT (Tyr220).
Antibodies in kit are custom formulations specific to kit
NOTE: Prepare solutions with purified water.
*NOTE: Some PathScan® ELISA Kits may include HRP-Linked Streptavidin in place of HRP-Linked Antibody.
NOTE: Initial color of positive reaction is blue, which changes to yellow upon addition of STOP Solution.
posted November 2013
Protocol Id: 204
CST's PathScan® Phospho-LAT (Tyr220) Sandwich ELISA Kit detects endogenous levels of Phospho-LAT when phosphorylated at Tyr191. As shown in Figure 1, a significant induction of LAT phosphorylation at Tyr191 can be detected in Jurkat cells following treatment with anti-CD3 using the Phospho-LAT (Tyr220) Sandwich ELISA Kit. The level of total LAT remains unchanged as shown by western analysis (Figure 1). This kit detects proteins from the indicated species, as determined through in-house testing, but may also detect homologous proteins from other species.
LAT, a transmembrane adaptor protein expressed in T, NK and mast cells, is an important mediator for T cell receptor (TCR) signaling (1). Upon TCR engagement, activated Zap-70 phosphorylates LAT at multiple conserved tyrosine residues within SH2 binding motifs, exposing these motifs as the docking sites for downstream signaling targets (2,3). The phosphorylation of LAT at Tyr171 and Tyr220 enables the binding of Grb2, Gads/SLP-76, PLCγ1 and PI3 kinase through their SH2 domain and translocates them to the membrane. This process eventually leads to activation of the corresponding signaling pathways (1-4).
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