ArticleJournal of proteome research2009
Two-dimensional southwestern blotting and characterization of transcription factors on-blot.
Article in Journal of proteome research, 2009. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 13 papers.
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Who cites it
13 citing papers in PubMed.
- Entropy-driven electrochemiluminescence ultra-sensitive detection strategy of NF-κB p50 as the regulator of cytokine storm.Biosensors & bioelectronics · 2021Article
- Method for trapping affinity chromatography of transcription factors using aldehyde-hydrazide coupling to agarose.Analytical biochemistry · 2015Article
- Southwestern Blotting Assay.Methods in molecular biology (Clifton, N.J.) · 2015Article
- Promoter trapping method: transcription factor purification using human telomerase reverse transcriptase promoter.Proteome science · 2014Article
- Asymmetric polymerase chain reaction provides alternatives for preparation of (GT)₅-tailed duplex DNA promoter for promoter trapping.Analytical biochemistry · 2012Article
- DNA-protein interactions: methods for detection and analysis.Molecular and cellular biochemistry · 2012Review
- Egr-1 induces DARPP-32 expression in striatal medium spiny neurons via a conserved intragenic element.The Journal of neuroscience : the official journal of the Society for Neuroscience · 2012Article
- Transcription factor proteomics: identification by a novel gel mobility shift-three-dimensional electrophoresis method coupled with southwestern blot and high-performance liquid chromatography-electrospray-mass spectrometry analysis.Journal of chromatography. A · 2011Article
- CCR5 promoter haplotype transcription complex characterization.Journal of health care for the poor and underserved · 2011Article
- Repeated probing of Southwestern blots using alkaline phosphatase stripping.Journal of chromatography. A · 2010Article
- Article
- Methods for proteomic analysis of transcription factors.Journal of chromatography. A · 2009Review
- Purification and characterization of transcription factors.Mass spectrometry reviewsReview
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Authors and funding
4 authors.
Funding
Abstract
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 microg of HEK293 nuclear extract without any previous purification.
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