Use of benzyldimethyl-n-hexadecylammonium chloride (“16-BAC”), a cationic detergent, in an acidic polyacrylamide gel electrophoresis system to detect base labile protein methylation in intact cells

Macfarlane, Donald E.

doi:10.1016/0003-2697(83)90001-5

Cited by 69 publications

(41 citation statements)

References 14 publications

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“…In addition to the mass spectrometry-based quantitation we used a gel-based strategy and combined the DIGE minimal labeling (18) of membrane proteins with a subsequent separation on 16-BAC/SDS two-dimensional gel (44,45). This approach allows the quantitation on the protein level and is well suited to analyze the dynamics of the proteome on a global scale as it is independent of mass spectrometric identification of individual gel spots, which may be a cumbersome task for integral membrane proteins.…”

Section: Discussionmentioning

confidence: 99%

“…After 1:1 dilution with a modified sample buffer (55 mM DTT, 1% (w/v) 16-BAC, 1% (v/v) glycerol, 0.05% Pyronin Y) proteins originating from both conditions were co-separated on a single gel. The first dimension was 12% 16-BAC-PAGE as described previously (44,45 Image Acquisition and Data Analysis-DIGE gels were scanned within the gel cassettes using Typhoon TM imager series (GE Healthcare) to prevent any changes in the dimensions or gel cracking during large format gel handling. Cy3 was excited by green (532 nm) and Cy5 was excited by red (633 nm) laser using appropriate emission filters (Cy3, 580 band pass 30; Cy5, 670 band pass 30) to minimize crosstalk.…”

Section: Methodsmentioning

confidence: 99%

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Quantitative Profiling of the Membrane Proteome in a Halophilic Archaeon

Bisle

Schmidt

Scheibe³

et al. 2006

Molecular & Cellular Proteomics

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We present a large scale quantitation study of the membrane proteome from Halobacterium salinarum. To overcome problems generally encountered with membrane proteins, we established a membrane preparation protocol that allows the application of most proteomic techniques originally developed for soluble proteins. Proteins were quantified using two complementary approaches. For gel-based quantitation, DIGE labeling was combined with two-dimensional gel electrophoresis on an improved 16-benzyldimethyl-n-hexadecylammonium chloride/SDS system. MS-based quantitation was carried out by combining gel-free separation with the recently developed isotope-coded protein labeling technique. Good correlations between these two independent quantitation strategies were obtained. From computational analysis we conclude that labeling of free amino groups by isotopecoded protein labeling (Lys and free N termini) is better suited for membrane proteins than Cys-based labeling strategies but that quantitation of integral membrane proteins remains cumbersome compared with soluble proteins. Nevertheless we could quantify 155 membrane proteins; 101 of these had transmembrane domains. We compared two growth states that strongly affect the energy supply of the cells: aerobic versus anaerobic/phototrophic conditions. The photosynthetic protein bacteriorhodopsin is the most highly regulated protein. As expected, several other membrane proteins involved in aerobic or anaerobic energy metabolism were found to be regulated, but in total, however, the number of regulated proteins is rather small. Molecular & Cellular Proteomics 5:1543-1558, 2006.

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Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Quantitative Profiling of the Membrane Proteome in a Halophilic Archaeon

Bisle

Schmidt

Scheibe³

et al. 2006

Molecular & Cellular Proteomics

View full text Add to dashboard Cite

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“…Nonetheless, it may be possible to analyze some membrane proteins by treating a membrane preparation with proteases, thus creating peptides, then separating these peptides by SDS-PAGE or LC [135]. Additionally, an alternative includes the use of 2-DE systems based solely on relative mobility [136,137]. In this case, there is no need to go through the pI (minimum of solubility), and very efficient, ionic detergents can be used throughout the procedure.…”

Section: Future Prospectsmentioning

confidence: 99%

Membrane proteins and proteomics: Un amour impossible?

2000

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“…To preserve the methyl esters formed on L-isoaspartyl residues, proteins were separated by 16-BAC acidic gel electrophoresis as described previously with minor modifications (Macfarlane 1983). Electrophoresis was performed overnight in 1.5-mm thick, 9% polyacrylamide, 3 M urea gels at a constant current of 50 mA.…”

Section: -Bac Acidic Gel Electrophoresismentioning

confidence: 99%

Down‐regulation of protein l‐isoaspartyl methyltransferase in human epileptic hippocampus contributes to generation of damaged tubulin

Lanthier

Bouthillier

Lapointe

et al. 2002

Journal of Neurochemistry

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Protein L-isoaspartyl methyltransferase (PIMT) repairs the damaged proteins which have accumulated abnormal aspartyl residues during cell aging. Gene targeting has elucidated a physiological role for PIMT by showing that mice lacking PIMT died prematurely from fatal epileptic seizures. Here we investigated the role of PIMT in human mesial temporal lobe epilepsy. Using surgical specimens of hippocampus and neocortex from controls and epileptic patients, we showed that PIMT activity and expression were 50% lower in epileptic hippocampus than in controls but were unchanged in neocortex. Although the protein was down-regulated, PIMT mRNA expression was unchanged in epileptic hippocampus, suggesting post-translational regulation of the PIMT level. Moreover, several proteins with abnormal aspartyl residues accumulate in epileptic hippocampus. Microtubules component b-tubulin, one of the major PIMT substrates, had an increased amount (two-fold) of L-isoaspartyl residues in the epileptic hippocampus. These results demonstrate that the down-regulation of PIMT in epileptic hippocampus leads to a significant accumulation of damaged tubulin that could contribute to neuron dysfunction in human mesial temporal lobe epilepsy.

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Use of benzyldimethyl-n-hexadecylammonium chloride (“16-BAC”), a cationic detergent, in an acidic polyacrylamide gel electrophoresis system to detect base labile protein methylation in intact cells

Cited by 69 publications

References 14 publications

Quantitative Profiling of the Membrane Proteome in a Halophilic Archaeon

Quantitative Profiling of the Membrane Proteome in a Halophilic Archaeon

Membrane proteins and proteomics: Un amour impossible?

Down‐regulation of protein l‐isoaspartyl methyltransferase in human epileptic hippocampus contributes to generation of damaged tubulin

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