Two-Dimensional Southwestern Blotting and Characterization of Transcription Factors On-Blot

Two-dimensional Southwestern blotting (2D-SW) described here combines several steps. Proteins are separated by two-dimensional gel electrophoresis and transferred to nitrocellulose (NC) or polyvinylidene fluoride (PVDF) membrane. The blotted proteins are then partially renatured and probed with a specific radiolabeled oligonucleotide for Southwestern blotting (SW) analysis. The detected proteins are then processed by on-blot digestion and identified by LC-MS/MS analysis. A transcription factor, bound by a specific radiolabeled element, is thus characterized without aligning with protein spots on a gel. In this study, we systematically optimize conditions for 2D-SW and on-blot digestion. By quantifying the SW signal using a scintillation counter, the optimal conditions for SW were determined to be PVDF membrane, 0.5% PVP40 for membrane blocking, serial dilution of guanidine HCl for denaturing and renaturing proteins on the blot, and an SDS stripping buffer to remove radiation from the blot. By the quantification of the peptide yields using nano-ESI-MS analysis, the optimized conditions for on-blot digestions were found to be 0.5% Zwittergent 3−16 and 30% acetonitrile in trypsin digestion buffer. With the use of the optimized 2D-SW technique and on-blot digestion combined with HPLC-nano-ESI-MS/MS, a GFP-C/EBP model protein was successfully characterized from a bacterial extract, and native C/EBP beta was identified from 100 μg of HEK293 nuclear extract without any previous purification.