title: Phos-tag SDS-PAGE systems for phosphorylation profiling of proteins with a wide range of molecular masses under neutral pH conditions.
abstract: We have previously reported a neutral-pH gel system buffered with Bis-Tris hydrochloride (Bis-Tris-HCl) in Zn(2+)-Phos-tag SDS-PAGE for advanced profiling of phosphoproteins with molecular masses of 10-200 kDa. In the current work, we describe characteristics of two neutral-pH gel systems, Bis-Tris-HCl and Tris-acetic acid (Tris-AcOH), based on comparative studies of the separation of a wide range of proteins with molecular masses from 10 to 350 kDa. For 10-200 kDa cellular proteins, the Bis-Tris-HCl system showed a higher resolving power in a 2-D fluorescence DIGE analysis of certain phosphoproteins, e.g. histone H3 (15 kDa) and elongation factor 2 (95 kDa). Furthermore, there was a large difference in the 1-D migration patterns of phosphorylated species of extracellular signal-regulated kinases 1 and 2 (ERK1/2, 44/42 kDa), which arise from changes in the phosphorylation status of the Thr-202 and Tyr-204, in the two buffer systems at the same concentration of Zn(2+)-Phos-tag. In contrast, shifts in the mobility of various phosphorylated species of a high-molecular-mass protein, ataxia telangiectasia-mutated kinase (ATM, 350 kDa), could only be detected in the Tris-AcOH system with a 3% w/v polyacrylamide gel strengthened with 0.5% w/v agarose.
authors: Department of Functional Molecular Science, Graduate School of Biomedical Sciences, Hiroshima University, Hiroshima, Japan. kinoeiji@hiroshima-u.ac.jp
authors: Eiji
authors: E
authors: Kinoshita
authors: Emiko
authors: E
authors: Kinoshita-Kikuta
authors: Tohru
authors: T
authors: Koike
mesh_terms: D000345:Affinity Labels / Q000737:chemistry; D064007:Ataxia Telangiectasia Mutated Proteins; D002021:Buffers; D018797:Cell Cycle Proteins / Q000737:chemistry*; D004268:DNA-Binding Proteins / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D024202:Electrophoretic Mobility Shift Assay / Q000379:methods; D006367:HeLa Cells; D006801:Humans; D006851:Hydrochloric Acid / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D008387:Marine Toxins; D019950:Mitogen-Activated Protein Kinase 1 / Q000737:chemistry; D048052:Mitogen-Activated Protein Kinase 3 / Q000737:chemistry; D008970:Molecular Weight; D010080:Oxazoles / Q000494:pharmacology; D010750:Phosphoproteins / Q000737:chemistry*; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry*; D025521:Tumor Suppressor Proteins / Q000737:chemistry*; D014746:Vimentin / Q000737:chemistry; D015032:Zinc / Q000737:chemistry
chemical_list: D000345:Affinity Labels; D002021:Buffers; D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D008387:Marine Toxins; D010080:Oxazoles; D010750:Phosphoproteins; D025521:Tumor Suppressor Proteins; D014746:Vimentin; C059041:calyculin A; C576324:ATM protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; D017346:Protein Serine-Threonine Kinases; C535150:MAPK1 protein, human; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D015032:Zinc; D006851:Hydrochloric Acid