Cell Signaling Technology

Product Pathways - PathScan ELISA

PathScan® Total Src Sandwich ELISA Kit #7984

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Kit Includes Volume Solution Color
Src Rabbit Antibody Coated Microwells 96 tests
Src 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 (10X), which is stored at –20°C (packaged separately).

Species Cross-Reactivity

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

Description

The PathScan® Total Src Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of total Src protein. A Src rabbit antibody has been coated onto the microwells. After incubation with cell lysates, Src protein (phospho and nonphospho) is captured by the coated antibody. Following extensive washing, a Src mouse detection antibody is added to detect the captured Src protein. Anti-mouse IgG, 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 total Src protein.

Specificity / Sensitivity

The PathScan® Total Src Sandwich ELISA Kit #7984 from Cell Signaling Technology detects endogenous levels of total Src protein, 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.

ELISA

ELISA

Figure 1. Constitutive phosphorylation of Src at Tyr416 in Calu-3 cells as detected by the PathScan® Phospho-Src (Tyr416) Sandwich ELISA Kit #7953. Treatment of lysate with λ-phosphatase significantly reduces Src phosphorylation but does not alter the levels of total Src protein as detected by the PathScan® Total Src Sandwich ELISA kit #7984. Absorbance at 450 nm is shown in the top figure while the corresponding western blots using Src (L4A1) Mouse mAb #2110 (left) or Phospho-Src Family (Tyr416) (100F9) Rabbit mAb #2113 (right) are shown in the bottom figure.

ELISA

ELISA

Figure 2. The relationship between lysate protein concentration from untreated and λ-phosphatase treated lysate from Calu-3 cells and the absorbance at 450 nm is shown.

Background

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).

  1. Thomas, S.M. and Brugge, J.S. (1997) Annu. Rev. Cell Dev. Biol. 13, 513-609.
  2. Hunter, T. (1987) Cell 49, 1-4.

Application References

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Companion Products

This product is intended for research purposes only. The product is not intended to be used for therapeutic or diagnostic purposes in humans or animals.

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