Figure 1. Treatment of Jurkat cells with H202 stimulates phosphorylation of Lck at Tyr505 as detected by the PathScan® Phospho-Lck (Tyr505) Sandwich ELISA Kit #7941, but does not affect the level of total Lck. Jurkat cells were treated with 20 mM H202 for 3 minutes at 37ºC. In untreated lysates, λ-phosphatase was used to abolish basal phosphorylation. The absorbance readings at 450 nm are shown in the top figure, while the western blots, using Lck (L22B1) Mouse mAb #2657 (lower panel) or Phospho-Lck (Tyr505) Antibody #2751 (upper panel), are shown in the bottom figure.
Figure 2. The relationship between lysate protein concentration from untreated and H202-treated Jurkat cells and the absorbance at 450 nm using PathScan® Phospho-Lck (Tyr505) Sandwich ELISA Kit #7941 is shown. Jurkat cells were treated with H202 (20 mM) for 3 minutes at 37ºC and then lysed.
|Product Includes||Volume||Solution Color|
|Phospho-Lck (Tyr505) Rabbit Antibody||1 ea|
|Lck 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|
|ELISA Sample Diluent||25 ml||Blue|
|TMB Substrate 7004||11 ml|
|STOP Solution 7002||11 ml|
|Sealing Tape||2 ea|
|ELISA Wash Buffer (20X) 9801||25 ml|
|Cell Lysis Buffer (10X) 9803||15 ml|
The PathScan® Phospho-Lck (Tyr505) Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of phospho-Lck (Tyr505). A phospho-Lck rabbit antibody has been coated onto the microwells. After incubation with cell lysates, phospho-Lck (Tyr505) is captured by the coated antibody. Following extensive washing, a Lck mouse detection mAb is added to detect the captured phospho-Lck (Tyr505). 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-Lck (Tyr505).
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-Lck (Tyr505) Sandwich ELISA Kit from Cell Signaling Technology detects endogenous levels of Lck when phosphorylated at Tyr505, as shown in Figure 1. This kit does not cross-react with the Src family members Yes, Src, 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.
Lck belongs to the Src-like non-receptor tyrosine kinase family with the typical Src family kinase structure: a unique amino terminal domain (Src homology 4 domain, SH4) followed by an SH3 domain, an SH2 domain, a kinase domain (SH1), and a carboxy-terminal negative regulatory domain (1). Lck activity is controlled by the interactions of SH2 and SH3 domains as well as tyrosine phosphorylation status of the activation loop (2,3). Lck is recruited to the T cell receptor (TCR) complex upon stimulation and activates downstream tyrosine kinases to initiate T cell signaling (4). Lck is also found to be involved in the regulation of mitochondrial apoptosis pathways and may be responsible for some anticancer drug induced apoptosis (5,6).
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