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[DESCRIPTION]
<p>KIRA-ELISA is used for quantitave detection of phosphorylation level of tyrosine kinase receptors. The protocol could be used for many different receptors. In our work, this protocol has been successful for TrkB and FGFR1 receptors. The underlying principle of the assay is capturing the receptor and detecting phosphorylated tyrosine on the intracellular domain of the receptor using pan phospho-tyrosine antibody. Since tyrosine kinase receptor are phosphorylated at multiple tyrosine residues, pan phospho-tyrosine antibody detects any possible antigen on the receptor, resulting in more sensitive assay. Our assay is based on previously published bioassay by M. Sadick et. al. and M. Cazorla et. al.</p>

[GUIDELINES]
<p><strong>Solutions and Buffers: </strong><br /><strong>Note:</strong> Do not use sodium azide in any buffers or solutions, as sodium azide inactivates the horseradish-peroxidase enzyme. <br /><br /><strong>Phosphate Buffered Saline (PBS): </strong><br />80.0 g NaCl <br />14.4 g Na2HPO4 <br />2.4 g KH2PO4 <br />2.0 g KCl <br />Add ddH2O up to 10 L, pH to 7.2 with HCl <br /><br /><strong>PBS/Tween: </strong><br />0.5 ml of Tween-20 in 1 L PBS<br /><br /> <strong>Blocking Solution: </strong><br />10% fetal bovine serum or 1% BSA in PBS. Filter before use to remove particulates.<br /><br /><strong>General References: </strong><br />1. Davies, C. 1994. The Immunoassay Handbook. D. Wild, Ed. Stockton Press, New York. <br />2. Abrams, J.S. 1995. Immunoenzymetric assay of mouse and human cytokines using NIP-labeled anti-cytokine antibodies. Current Protocols in Immunology (J. Coligan, A. Kruisbeek, D. Margulies, E. Shevach, W. Strober, Eds). John Wiley and Sons, New York. Unit 6.20. <br />3. Sander, B., et al. 1993. J. Immunol. Meth. 166:201. <br />4. Abrams, J.S., et al. 1992. Immunol. Rev. 127:5. <br /><br /><strong>Cytokine ELISA Troubleshooting Tips <br /><br />Poor signal-to-noise ratio </strong><br />• Try Capture Antibody at 1 – 10 µg/ml (generally 2 µg/ml). <br />• Try Detection Antibody at 0.25 – 2 µg/ml (generally 1 µg/ml). <br />• Titrate against each other to obtain optimal dilutions. <br /><br /><strong>Low Sensitivity </strong><br />• Try overnight incubations of standards and samples at 4°C.<br /><br /><strong>Poor Signal </strong><br />• If using HRP, avoid sodium azide in wash buffers and diluents, as sodium azide inhibits HRP. <br />• Verify that appropriate antibody pairs were used and the activity of the samples and/or standards. <br />• Check the activity of enzyme and substrate by coating with Detection Antibody (1 µg/ml), adding biotinylated avidin and revealing with the appropriate substrate. If the enzyme/substrate is active, a strong signal should be observed. <br /><br /><strong>Poor Standard Curve </strong><br />• Handling Instructions for standards are lot-specific. Refer to product information for proper handling. <br />• Recombinant protein vials should be quick-spun for maximum recovery. <br />• BioLegend suggests that cytokines be stored in a concentrated format (&gt;100 ng/ml) and in the presence of a protein carrier. <br /><br /><strong>High Background </strong><br />• Increase stringency of washing steps by soaking plates for ~1 minute during washes. <br />• Determine optimum Capture and Detection Antibody dilutions. <br />• Increase the dilution of Detection Antibody and/or increase the number of washes after Av-HRP incubation.</p>

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