Figure 2. The relationship between lysate protein concentration from untreated and H202-treated MDCK cells and the absorbance at 450 nm is shown. MDCK cells were treated with H202 (3 mM) for 3 minutes at 37ºC and then lysed.
Figure 1. Phosphorylation of Src at Tyr416 in H202 stimulated MDCK cells (3 mM for 3 min) and untreated Calu-3 cells is detected by the PathScan® Phospho-Src (Tyr416) Sandwich ELISA Kit. In contrast, a low level of phospho-Src (Tyr416) is detected in untreated MDCK cells and Calu-3 cells lysed in the presence of λ-phosphatase. The absorbance readings at 450 nm are shown in the top figure while western blots, using Phospho-Src Family (Tyr416) (100F9) Rabbit mAb #2113 (upper) or Src (L4A1) Mouse mAb #2110 (lower), are shown in the bottom figure.
|Product Includes||Volume||Solution Color|
|Phospho-Src (Tyr416) Rabbit mAb Coated Microwells||96 tests|
|Src Mouse Detection mAb||1 ea||Green (Lyophilized)|
|Anti-mouse 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-Src (Tyr416) Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of phospho-Src (Tyr416). A phospho-Src rabbit antibody has been coated onto the microwells. After incubation with cell lysates, phospho-Src (Tyr416) is captured by the coated antibody. Following extensive washing, a Src mouse detection antibody is added to detect the captured phospho-Src (Tyr416). Anti-mouse, HRP-linked 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-Src (Tyr416).
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
The PathScan® Phospho-Src (Tyr416) Sandwich ELISA Kit from Cell Signaling Technology detects endogenous levels of Src when phosphorylated at Tyr416, as shown in Figure 1. This kit does not cross-react with the Src family members Yes, Lck, Lyn or Hck. Other family members have not been tested. The kit sensitivity is shown in Figure 2. This kit detects proteins from the indicated species, as determined through in-house testing, but may also detect homologous proteins from other species.
The Src family of protein tyrosine kinases, which includes Src, Lyn, Fyn, Yes, Lck, Blk, and Hck, are important in the regulation of growth and differentiation of eukaryotic cells (1). Src activity is regulated by tyrosine phosphorylation at two sites, but with opposing effects. While phosphorylation at Tyr416 in the activation loop of the kinase domain upregulates enzyme activity, phosphorylation at Tyr527 in the carboxy-terminal tail by Csk renders the enzyme less active (2).
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