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

Abstract

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.

Detection of Activated Receptor Tyrosine Kinases in Human Lung Squamous Cell Carcinoma

“…We used a sensitive method of pTyr WB [48] to analyze homogenates of resected tumor tissue from 12 LSCC, 2 LADC, and 11 lung controls for the presence of RTKs activated by tyrosine transphosphorylation. Since RTKs are known to be high MW proteins, we used this parameter as the initial criterion for a positive result.…”

Integrative multiomic analyses of dorsal root ganglia in diabetic neuropathic pain using proteomics, phospho-proteomics, and metabolomics

“…Two-dimensional electrophoresis was performed with 500 ug of protein by Kendrick Labs, Inc. (Madison, WI) according to the carrier ampholine method of isoelectric focusing 67 . Isoelectric focusing was carried out in a glass tube of inner diameter 3.3 mm using 2.0% pH 3–10 isodalt Servalytes (Serva, Heidelberg, Germany) for 20,000 V-h. Tube gels were then sealed to the top of a stacking gel that overlaid a 10% acrylamide slab gel (1.0 mm) and SDS slab gel electrophoresis was carried out.…”

Integrative Multiomic Analyses of Diabetic Neuropathic Pain in Dorsal Root Ganglia: Proteomics, Phospho-proteomics, and Metabolomics

“…Two-dimensional electrophoresis was performed with 500 ug of protein by Kendrick Labs, Inc. (Madison, WI) according to the carrier ampholine method of isoelectric focusing [66]. Isoelectric focusing was carried out in a glass tube of inner diameter 3.3 mm using 2.0% pH 3-10 isodalt Servalytes (Serva, Heidelberg, Germany) for 20,000 volt-hrs.…”

“…Two-dimensional electrophoresis was performed with 500 ug of protein by Kendrick Labs, Inc. (Madison, WI) according to the carrier ampholine method of isoelectric focusing [66].…”