Preparation of a phosphotyrosine-protein standard for use in semiquantitative western blotting with enhanced chemiluminescence

• Published in: PloS one Vol. 15; no. 6; p. e0234645
• Main Authors: Kendrick, Nancy, Powers, Ginny, Johansen, Jon, Hoelter, Matt, Koll, Andrew, Carlson, Sofia, Channaveerappa, Devika, Darie, Costel C
• Format: Journal Article
• Language: English
• Published: San Francisco Public Library of Science 18.06.2020; Public Library of Science (PLoS)
• Subjects: Antibodies; Biochemistry; Biology and Life Sciences; Carcinogenesis; Chemiluminescence; Density; Epidermal growth factor; Epidermal growth factor receptors; Feasibility studies; Gene expression; Genetic aspects; Growth factors; Health aspects; Image detection; Kinases; Linearity; Lymphoma; Lysates; Medicine and Health Sciences; Molecular weight; Phosphorylation; Phosphotransferases; Phosphotyrosine; Physical Sciences; Protein research; Protein-tyrosine kinase; Proteins; Proteomics; Receptors; Research and Analysis Methods; Signal transduction; Tumors; Tyrosine; Western blotting; United States; United States > US
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0234645

Protein tyrosine phosphorylation is key to activation of receptor tyrosine kinases (RTK) that drive development of some cancers. One challenge of RTK-targeted therapy is identification of those tumors that express non-mutated but activated RTKs. Phosphotyrosine (pTyr) RTK levels should be more predictive of the latter than expressed total protein. Western blotting (WB) with a pTyr antibody and enhanced chemiluminescence (ECL) detection is sufficiently sensitive to detect pTyr-RTKs in human tumor homogenates. Presentation of results by comparing WB images, however, is wanting. Here we describe the preparation of a new pTyr-protein standard, pTyr-ALK48-SB (pA), derived from a commercial anaplastic lymphoma kinase (ALK) recombinant fragment, and its use to quantify pTyr-epidermal growth factor receptor (pTyr-EGFR) in commercial A431 cell lysates. Linearity of one-dimensional (1D) WB plots of pA band density versus load as well as its lower level of detection (0.1 ng, 2 fmole) were determined for standardized conditions. Adding pA to two lots of A431 cell lysates with high and low pTyr-EGFR allowed normalization and quantification of the latter by expressing results as density ratios for both 1D and 2D WB. This approach is semi-quantitative because unknown RTKs may be outside the linear range of detection. Semiquantitative ratios are an improvement over comparisons of images without a reference standard and facilitate comparisons between samples.