LIF detection of peptides and proteins in CE

Garcı́a-Campaña, Ana M.; Taverna, Myriam; Fabre, H.

doi:10.1002/elps.200500790

Cited by 91 publications

(79 citation statements)

References 172 publications

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“…An important application of deep UV fluorescence is the detection of unlabelled proteins [13,28] where tryptophan, tyrosine and marginally phenylalanine act as native fluorophores. We could previously show that underivatised proteins can be detected at low micromolar concentrations in chip electrophoresis utilising deep UV fluorescence detection [12].…”

Section: Detection Of Proteinsmentioning

confidence: 99%

Impact of laser excitation intensity on deep UV fluorescence detection in microchip electrophoresis

2008

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A high intensity 266 nm continuous wave (cw-) laser developed for material processing was utilised as an excitation source for sensitive native fluorescence detection of unlabelled compounds in MCE. This 120 mW laser was attached via an optical fibre into a commercial epifluorescence microscope. With this MCE set-up we evaluated the impact of laser power on the S/N of aromatic compounds as well as of proteins. Compared with a previous work which used a 4 mW pulsed laser for excitation, improved S/N for small aromatics and to a lesser extent for proteins could be attained. The LOD of the system was determined down to 24 ng/mL for serotonin (113 nM), 24 ng/mL for propranolol (81 nM), 80 ng/mL for tryptophan (392 nM) and 80 ng/mL for an aromatic diol (475 nM). Sensitive protein detection was obtained at concentrations of 5 mg/mL for lysocyme, trypsinogen and chymotrypsinogen (340, 208 and 195 nM, respectively). Finally, a comparison of the cw-with a pulsed 266 nm laser, operating at the same average power, showed a higher attainable sensitivity of the cw-laser. This can be attributed to fluorescence saturation and photobleaching effects of the pulsed laser at high pulse energies.

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Section: Detection Of Proteinsmentioning

confidence: 99%

Impact of laser excitation intensity on deep UV fluorescence detection in microchip electrophoresis

2008

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“…In CE, proteins are typically detected using UV absorption, laser induced fluorescence (LIF), or by coupling to a mass spectrometer (MS) (Fonslow & Yates, 2009;Garcia-Campana, Taverna, & Fabre, 2007;Herrero, Ibanez, & Cifuentes, 2008;Kasicka, 2008;Stutz, 2005). A limitation when using UV detection is that the limit of detection (LOD) is in the micromolar range.…”

Section: Using Capillary Electrophoresis To Identify the Proteome Of mentioning

confidence: 99%

Proteomics Analysis of Kinetically Stable Proteins

Xia¹,

Manning²,

Zhang³

et al. 2012

Integrative Proteomics

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“…The recombinant plasmid pHis 6 -Erk1-TC was constructed from the Erk1 plasmid pETHis 6 /MEK1R4F+ERK1 (a kind gift from Dr. Melanie Cobb). 20 Codons encoding the His 6 tag and the TC motif LNCCPGCCMEP were introduced into the 5'and 3' ends, respectively, of the original plasmid by standard polymerase chain reaction (PCR).…”

Section: Construction Of Erk1-tcmentioning

confidence: 99%

Biarsenical−Tetracysteine Motif as a Fluorescent Tag for Detection in Capillary Electrophoresis

et al. 2008

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Biarsenical dyes complexed to tetracysteine motifs have proven to be highly useful fluorescent dyes in labeling specific cellular proteins for microscopic imaging. Their many advantages include membrane permeability, relatively small size, stoichiometric labeling, high affinity, and an assortment of excitation/emission wavelengths. The goal of the current study was to determine whether the biarsenical labeling scheme could be extended to fluorescent detection of analytes in capillary electrophoresis. Recombinant protein or synthesized peptides containing the optimized tetracysteine motif "-C-C-P-G-C-C-" were labeled with biarsenical dyes and then analyzed by MEKC. The biarsenical-tetracysteine complex was stable and remained fluorescent under standard micellar electrokinetic capillary chromatography (MEKC) conditions for peptide and protein separations. The detection limit following electrophoresis in a capillary was less than 3 × 10 −20 moles with a simple laser-induced fluorescence system. A mixture of multiple biarsenical-labeled peptides and a protein were easily resolved. Demonstrating that the label did not interfere with bioactivity, a peptide-based enzyme substrate conjugated to the tetracysteine motif and labeled with a biarsenical dye retained its ability to be phosphorylated by the parent kinase. The feasibility of using this label for chemical cytometry experiments was shown by intracellular labeling and subsequent analysis of a recombinant protein possessing the tetracysteine motif expressed in living cells. The extension of the biarsenical-tetracysteine tag to fluorescent labeling of peptides and proteins in chemical separations is a valuable addition to biochemical and cell-based investigations.

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LIF detection of peptides and proteins in CE

Cited by 91 publications

References 172 publications

Impact of laser excitation intensity on deep UV fluorescence detection in microchip electrophoresis

Impact of laser excitation intensity on deep UV fluorescence detection in microchip electrophoresis

Proteomics Analysis of Kinetically Stable Proteins

Biarsenical−Tetracysteine Motif as a Fluorescent Tag for Detection in Capillary Electrophoresis

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