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REACTIVITY
H
Product Includes Volume Solution Color
TrkB Mouse mAb coated microwells 96 tests
Biotinylated P-Tyrosine Detection Ab 5.5 ml Green
HRP-Linked Streptavidin 5.5 ml Red
Luminol/Enhancer Solution 3 ml Colorless
Stable Peroxide Buffer 3 ml Colorless
Sealing Tape 2 sheets
ELISA Wash Buffer (20X) 25 ml Colorless
ELISA Sample Diluent 25 ml Blue
Cell Lysis Buffer (10X) 9803 15 ml Yellowish

Order Details

Custom Ordering Details: When ordering five or more kits, please contact us for processing time and pricing at sales@cellsignal.com.
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ELISA Chemiluminescent

NOTE: Refer to product-specific datasheets or product webpage for assay incubation temperature. This /product/productDetail.jsp?productId=luminescent ELISA is offered in low volume microplate. Samples and reagents only require 50 µl per microwell.

A. Solutions and Reagents

NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.

  1. 20X Phosphate Buffered Saline (PBS): (#9808) To prepare 1 L 1X PBS: add 50 ml 10X PBS to 950 ml dH2O, mix.
  2. Bring all microwell strips to room temperature before use.
  3. Prepare 1X wash buffer by diluting 20X Wash Buffer (included in each PathScan® Sandwich ELISA Kit) in dH2O.
  4. 1X Cell Lysis Buffer: 10X Cell Lysis Buffer (#9803): To prepare 10 ml of 1X Cell Lysis Buffer, add 1 ml of 10X Cell Lysis Buffer to 9 ml of dH2O, mix. Buffer can be stored at 4°C for short-term use (1–2 weeks).

    Recommended: Add 1 mM phenylmethylsulfonyl fluoride (PMSF) (#8553) immediately before use.

  5. 20X LumiGLO® Reagent and 20X Peroxide: (#7003).

B. Preparing Cell Lysates

For adherent cells

  1. Aspirate media when the culture reaches 80–90% confluence. Treat cells by adding fresh media containing regulator for desired time.
  2. Remove media and rinse cells once with ice-cold 1X PBS.
  3. Remove PBS and add 0.5 ml ice-cold 1X Cell Lysis Buffer plus 1 mM PMSF to each plate (10 cm diameter) and incubate the plate on ice for 5 min.
  4. Scrape cells off the plate and transfer to an appropriate tube. Keep on ice.
  5. Sonicate lysates on ice.
  6. Microcentrifuge for 10 min (x14,000 rpm) at 4°C and transfer the supernatant to a new tube. The supernatant is the cell lysate. Store at -80°C in single-use aliquots.

For suspension cells

  1. Remove media by low speed centrifugation (~1,200 rpm) when the culture reaches 0.5–1.0 x 106 viable cells/ml. Treat cells by adding fresh media containing regulator for desired time.
  2. Collect cells by low speed centrifugation (~1,200 rpm) and wash once with 5–10 ml ice-cold 1X PBS.
  3. Cells harvested from 50 ml of growth medium can be lysed in 2.0 ml of 1X cell lysis buffer plus 1 mM PMSF.
  4. Sonicate lysates on ice.
  5. Microcentrifuge for 10 min (x14,000 rpm) at 4°C and transfer the supernatant to a new tube. The supernatant is the cell lysate. Store at -80°C in single-use aliquots.

C. Test Procedure

  1. After the microwell strips have reached room temperature, break off the required number of microwells. Place the microwells in the strip holder. Unused microwells must be resealed in the storage bag and stored at 4°C immediately.
  2. Cell lysates can be used undiluted or diluted with sample diluent (supplied in each PathScan® Sandwich ELISA Kit, blue color). Individual datasheets or product webpage for each kit provide information regarding an appropriate dilution factor for lysates and kit assay results.
  3. Add 50 µl of each undiluted or diluted cell lysate to the appropriate well. Seal with tape and press firmly onto top of microwells. Incubate the plate for 2 hr at room temperature. Alternatively, the plate can be incubated overnight at 4°C.
  4. Gently remove the tape and wash wells:
    1. Discard plate contents into a receptacle.
    2. Wash 4 times with 1X Wash Buffer, 150 µl each time per well.
    3. For each wash, strike plates on fresh paper towels hard enough to remove the residual solution in each well, but do not allow wells to dry completely at any time.
    4. Clean the underside of all wells with a lint-free tissue.
  5. Add 50 µl of detection antibody (green color) to each well. Seal with tape and incubate the plate at room temperature for 1 hr.
  6. Repeat wash procedure (Section C, Step 4).
  7. Add 50 µl of HRP-linked secondary antibody (red color) to each well. Seal with tape and incubate the plate at room temperature for 30 min.
  8. Repeat wash procedure (Section C, Step 4).
  9. Prepare detection reagent working solution by mixing equal parts 2X LumiGLO® Reagent and 2X Peroxide.
  10. Add 50 µl of the detection reagent working solution to each well.
  11. Use a plate-based luminometer set at 425 nm to measure Relative Light Units (RLU) within 1–10 min following addition of the substrate.
    1. Optimal signal intensity is achieved when read within 10 min.

posted November 2009

revised September 2013

protocol id: 41

Product Description

The PathScan® Phospho-TrkB (panTyr) Chemiluminescent Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of tyrosine-phosphorylated TrkB protein with a chemiluminescent readout. Chemiluminescent ELISAs often have a wider dynamic range and higher sensitivity than conventional chromogenic detection. This chemiluminescent ELISA, which is offered in low volume microplates, shows increased signal and sensitivity while using a smaller sample size. A TrkB Mouse mAb has been coated onto the microwells. After incubation with cell lysates, TrkB (phospho and nonphospho) is captured by the coated antibody. Following extensive washing, a Biotinylated Phospho-Tyrosine Detection Antibody is added to detect captured tyrosine-phosphorylated TrkB protein. HRP-linked Streptavidin is then used to recognize the bound detection antibody. Chemiluminescent reagent is added for signal development. The magnitude of light emission, measured in relative light units (RLU), is proportional to the quantity of tyrosine-phosphorylated TrkB protein.

Antibodies in kit are custom formulations specific to kit.


Specificity / Sensitivity

PathScan® Phospho-TrkB (panTyr) Chemiluminescent Sandwich ELISA Kit detects endogenous levels of TrkB protein when phosphorylated at Tyr residues in human cells. This kit detects proteins from the indicated species, as determined through in-house testing, but may also detect homologous proteins from other species.


Species Reactivity: Human

The family of Trk receptor tyrosine kinases consists of TrkA, TrkB, and TrkC. While the sequence of these family members is highly conserved, they are activated by different neurotrophins: TrkA by NGF, TrkB by BDNF or NT4, and TrkC by NT3 (1). Neurotrophin signaling through these receptors regulates a number of physiological processes, such as cell survival, proliferation, neural development, and axon and dendrite growth and patterning (1). In the adult nervous system, the Trk receptors regulate synaptic strength and plasticity. TrkA regulates proliferation and is important for development and maturation of the nervous system (2). Phosphorylation at Tyr490 is required for Shc association and activation of the Ras-MAP kinase cascade (3,4). Residues Tyr674/675 lie within the catalytic domain, and phosphorylation at these sites reflects TrkA kinase activity (3-6). Point mutations, deletions, and chromosomal rearrangements (chimeras) cause ligand-independent receptor dimerization and activation of TrkA (7-10). TrkA is activated in many malignancies including breast, ovarian, prostate, and thyroid carcinomas (8-13). Research studies suggest that expression of TrkA in neuroblastomas may be a good prognostic marker as TrkA signals growth arrest and differentiation of cells originating from the neural crest (10).


Many tyrosine phosphorylation sites are conserved between TrkA and TrkB: Tyr490 of TrkA corresponds to Tyr512 in TrkB, and Tyr674/675 of TrkA to Tyr706/707 in TrkB of the human sequence (14). TrkB is overexpressed in tumors such as neuroblastoma, prostate adenocarcinoma, and pancreatic ductal adenocarcinoma (15). Research studies have shown that in neuroblastomas, overexpression of TrkB correlates with an unfavorable disease outcome when autocrine loops signaling tumor survival are potentiated by additional overexpression of brain-derived neurotrophic factor (BDNF) (16-18). An alternatively spliced truncated TrkB isoform lacking the kinase domain is overexpressed in Wilms’ tumors and this isoform may act as a dominant-negative regulator of TrkB signaling (17).


1.  Huang, E.J. and Reichardt, L.F. (2003) Annu Rev Biochem 72, 609-42.

2.  Segal, R.A. and Greenberg, M.E. (1996) Annu Rev Neurosci 19, 463-89.

3.  Stephens, R.M. et al. (1994) Neuron 12, 691-705.

4.  Marsh, H.N. et al. (2003) J Cell Biol 163, 999-1010.

5.  Reuther, G. W. et al. (2000) Mol. Cell. Biol. 20, 8655-8666.

6.  Obermeier, A. et al. (1993) EMBO J 12, 933-41.

7.  Obermeier, A. et al. (1994) EMBO J 13, 1585-90.

8.  Arevalo, J.C. et al. (2001) Oncogene 20, 1229-34.

9.  Greco, A. et al. (1997) Genes Chromosomes Cancer 19, 112-23.

10.  Pierotti, M.A. and Greco, A. (2006) Cancer Lett 232, 90-8.

11.  Lagadec, C. et al. (2009) Oncogene 28, 1960-70.

12.  Greco, A. et al. (2010) Mol Cell Endocrinol 321, 44-9.

13.  Ødegaard, E. et al. (2007) Hum Pathol 38, 140-6.

14.  Huang, E.J. and Reichardt, L.F. (2003) Annu Rev Biochem 72, 609-42.

15.  Geiger, T.R. and Peeper, D.S. (2005) Cancer Res 65, 7033-6.

16.  Han, L. et al. (2007) Med Hypotheses 68, 407-9.

17.  Aoyama, M. et al. (2001) Cancer Lett 164, 51-60.

18.  Desmet, C.J. and Peeper, D.S. (2006) Cell Mol Life Sci 63, 755-9.


Entrez-Gene Id 4915
Swiss-Prot Acc. Q16620


For Research Use Only. Not For Use In Diagnostic Procedures.
Cell Signaling Technology® is a trademark of Cell Signaling Technology, Inc.
PathScan® is a trademark of Cell Signaling Technology, Inc.