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

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.

Correction to: 2D SDS PAGE in Combination with Western Blotting and Mass Spectrometry Is a Robust Method for Protein Analysis with Many Applications.

This chapter was previously published as non-open access. This is updated to an open access chapter now.

2D SDS PAGE in Combination with Western Blotting and Mass Spectrometry Is a Robust Method for Protein Analysis with Many Applications.

Two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (2D SDS PAGE) is a method that separates proteins according to their isoelectric points in the first dimension and molecular masses in the second dimension. Evidence is provided that 2D SDS PAGE is reproducible, robust and compatible with SDS in both dimensions including isoelectric focusing in tube gels, the first dimension. The 2D gel pattern of rat liver microsomes shows more detail and sharper spot outlines when dissolved in SDS buffer with heating than in urea buffer and is better yet when dissolved in a mixture of both buffers.