Photopolymerization of polyacrylamide gels with methylene blue

Lyubimova, Tatyana; Caglio, Silvia; Gelfi, Cecilia; Righetti, Pier Giorgio; Rabilloud, Thierry

doi:10.1002/elps.1150140108

Cited by 58 publications

(47 citation statements)

References 27 publications

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“…For polypeptides above 25 kDa, the best separation in the first dimension was observed when electrophoresis was carried out under strongly denaturing conditions at acidic pH in a gel matrix with large pores (i.e., when 2% acrylamide-1.4% methylene bisacrylamide was used). Under these conditions, polymerization could not be initiated with the commonly used TEMED-persulfate but instead required photopolymerization with methylene blue (23). The best results were obtained with gels containing 7 M urea, 4 M thiourea, and 40% (wt/vol) formamide.…”

Section: Vol 31 2011 Semiquantitative Proteomics Of Human Spliceosomentioning

confidence: 99%

Semiquantitative Proteomic Analysis of the Human Spliceosome via a Novel Two-Dimensional Gel Electrophoresis Method

Agafonov

Deckert

Wolf

et al. 2011

Molecular and Cellular Biology

197

304

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More than 200 proteins associate with human spliceosomes, but little is known about their relative abundances in a given spliceosomal complex. Here we describe a novel two-dimensional (2D) electrophoresis method that allows separation of high-molecular-mass proteins without in-gel precipitation and thus without loss of protein. Using this system coupled with mass spectrometry, we identified 171 proteins altogether on 2D maps of stage-specific spliceosomal complexes. By staining with a fluorescent dye with a wide linear intensity range, we could quantitate and categorize proteins as present in high, moderate, or low abundance. Affinitypurified human B, B act , and C complexes contained 69, 63, and 72 highly/moderately abundant proteins, respectively. The recruitment and release of spliceosomal proteins were followed based on their abundances in A, B, B act , and C spliceosomal complexes. Staining with a phospho-specific dye revealed that approximately one-third of the proteins detected in human spliceosomal complexes by 2D gel analyses are phosphorylated. The 2D gel electrophoresis system described here allows for the first time an objective view of the relative abundances of proteins present in a particular spliceosomal complex and also sheds additional light on the spliceosome's compositional dynamics and the phosphorylation status of spliceosomal proteins at specific stages of splicing.The spliceosome is a highly complex and dynamic megadalton RNP machine. It is comprised of the five snRNPs U1, U2, U4, U5, and U6 and a large number of non-snRNP protein factors (reviewed in reference 42). Spliceosomes assemble de novo in a stepwise manner on each new intron to be spliced and thus pass through a series of distinct complexes (42). Initially, the U1 snRNP binds the pre-mRNA, forming the E complex, and after stable U2 snRNP interaction, the A complex is generated. Subsequently, the U4/U6 and U5 snRNPs associate, as part of the U4/U6.U5 tri-snRNP, and the precatalytic B complex is formed. Through a series of compositional and structural rearrangements, the B complex is activated, first yielding the B act complex. After the action of the DEXH box protein Prp2, the B* complex is formed, which catalyzes step 1 of splicing. This involves cleavage at the 5Ј splice site (ss) of the pre-mRNA and the ligation of the 5Ј end of the intron to the so-called branch site to form a lariat-like structure. After the first step, the spliceosomal C complex is formed, and it catalyzes the step 2 of splicing, during which the intron is excised and the exons are ligated together to form mRNA.Mass spectrometry (MS) analyses have shown that more than 200 proteins copurify with mixtures of human spliceosomal complexes (31, 47). Individual spliceosomal complexes contain many fewer proteins (e.g., ϳ125 for B, B act , and C complexes) and differ from each other considerably in composition (3,6,7,10). However, the relative abundances of all of the proteins present within a given spliceosomal complex are presently not clear. Spliceosomes contain...

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Section: Vol 31 2011 Semiquantitative Proteomics Of Human Spliceosomentioning

confidence: 99%

Semiquantitative Proteomic Analysis of the Human Spliceosome via a Novel Two-Dimensional Gel Electrophoresis Method

Agafonov

Deckert

Wolf

et al. 2011

Molecular and Cellular Biology

197

304

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“…Gels were photopolymerized as described by Lyubimova et al (1993). SOD activity was visualized as described by Beauchamp and Fridovich (1971).…”

Section: Sod Activity Gels and Activity Assaysmentioning

confidence: 99%

Fe Sparing and Fe Recycling Contribute to Increased Superoxide Dismutase Capacity in Iron-Starved Chlamydomonas reinhardtii

et al. 2012

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“…First-dimension isoelectric focusing (IEF) was performed using tube gels in a Bio-Rad mini-IEF system. Due to the presence of thiourea in the sample and the gel, an alternative method to polymerize the gel was used, as thiourea interferes with the commonly used APS/ Temed method (27,30). Approximately 4 ϫ 10 6 cell equivalents were used for focusing, and a subsequent second-dimension SDS-PAGE was performed.…”

Section: Methodsmentioning

confidence: 99%

Uncovering a Role for the Tail of the Dictyostelium discoideum SadA Protein in Cell-Substrate Adhesion

Kowal¹,

Chisholm²

2011

Eukaryotic Cell

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Previous work from our laboratory showed that the Dictyostelium discoideum SadA protein plays a central role in cell-substrate adhesion. SadA null cells exhibit a loss of adhesion, a disrupted actin cytoskeleton, and a cytokinesis defect. How SadA mediates these phenotypes is unknown. This work addresses the mechanism of SadA function, demonstrating an important role for the C-terminal cytoplasmic tail in SadA function. We found that a SadA tailless mutant was unable to rescue the sadA adhesion deficiency, and overexpression of the SadA tail domain reduced adhesion in wild-type cells. We also show that SadA is closely associated with the actin cytoskeleton. Mutagenesis studies suggested that four serine residues in the tail, S924/S925 and S940/ S941, may regulate association of SadA with the actin cytoskeleton. Glutathione S-transferase pull-down assays identified at least one likely interaction partner of the SadA tail, cortexillin I, a known actin bundling protein.Thus, our data demonstrate an important role for the carboxy-terminal cytoplasmic tail in SadA function and strongly suggest that a phosphorylation event in this tail regulates an interaction with cortexillin I. Based on our data, we propose a model for the function of SadA.

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Photopolymerization of polyacrylamide gels with methylene blue

Cited by 58 publications

References 27 publications

Semiquantitative Proteomic Analysis of the Human Spliceosome via a Novel Two-Dimensional Gel Electrophoresis Method

Semiquantitative Proteomic Analysis of the Human Spliceosome via a Novel Two-Dimensional Gel Electrophoresis Method

Fe Sparing and Fe Recycling Contribute to Increased Superoxide Dismutase Capacity in Iron-Starved Chlamydomonas reinhardtii

Uncovering a Role for the Tail of the Dictyostelium discoideum SadA Protein in Cell-Substrate Adhesion

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