Cell Signaling Technology

Product Pathways - PathScan ELISA

PathScan® Phospho-Lck (Tyr505) Sandwich ELISA Kit #7941

When ordering five or more kits, please contact us for processing time and pricing at sales@cellsignal.com.

Important Ordering Details: Product is assembled upon order to ensure maximum activity. Domestic: Please allow up to two weeks for your order to be processed and shipped. International: Please allow up to three weeks, depending on the country, for your order to be processed and shipped.

Kit Includes Volume Solution Color
Phospho-Lck (Tyr505) Rabbit Antibody Coated Microwells 96 tests
Lck Mouse Detection Antibody 11 ml Green
Anti-mouse IgG, HRP-linked Antibody 11 ml Red
Sample Diluent 25 ml Blue
TMB Substrate #7004 11 ml Colorless
STOP Solution #7002 11 ml Colorless
Sealing Tape 2 sheets
ELISA Wash Buffer (20X) 25 ml Colorless
Cell Lysis Buffer (10X) #9803 15 ml Yellowish

Note: 12 8-well modules –Each module is designed to break apart for 8 tests.
Note: Kit should be stored at 4°C with the exception of Cell Lysis Buffer, which is stored at –20°C (packaged separately).

Species Cross-Reactivity

H

Reactivity Key:  H=Human
Species enclosed in parentheses are predicted to react based on 100% sequence homology.

Description

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).

Specificity / Sensitivity

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.

ELISA - Western correlation

ELISA - Western correlation

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.

Sensitivity

Sensitivity

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.

Background

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).

  1. Palacios, E.H. and Weiss, A. (2004) Oncogene 23, 7990-8000.
  2. Mustelin, T. and Taskén, K. (2003) Biochem J 371, 15-27.
  3. Gervais, F.G. et al. (1993) Mol Cell Biol 13, 7112-21.
  4. Straus, D.B. and Weiss, A. (1992) Cell 70, 585-93.
  5. Belka, C. et al. (2003) Oncogene 22, 176-85.
  6. Gruber, C. et al. (2004) Biochem Pharmacol 67, 1859-72.

Application References

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Companion Products

For Research Use Only. Not For Use In Diagnostic Procedures.

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