Capillary Electrophoresis for the Analysis of Biopolymers

Quigley, Wes W. C.; Dovic̀hi, Norman J.

doi:10.1021/ac040100d

Cited by 85 publications

(66 citation statements)

References 190 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…16 However, the addition of fluorescent labels to proteins can affect their ligand binding, solubility and stability, and it is also difficult to ensure the attachment of only a single label per protein molecule. 17,18 The measurement of intrinsic protein fluorescence has also been demonstrated previously in microchannels for the label-free detection of proteins and their conformational changes, by using lasers [19][20][21] or light-emitting diodes (LED's) 22,23 for the excitation of samples. However, these techniques have not yet been sufficiently accurate to obtain thermodynamic parameters such as the free-energy of protein unfolding, as is required for use in drug-discovery, formulation or protein engineering applications.…”

Section: Introductionmentioning

confidence: 99%

Protein denaturation and protein:drugs interactions from intrinsic protein fluorescence measurements at the nanolitre scale

et al. 2010

View full text Add to dashboard Cite

Protein stability and ligand-binding affinity measurements are widely required for the formulation of biopharmaceutical proteins, protein engineering and drug screening within life science research. Current techniques either consume too much of often precious biological or compound materials, in large sample volumes, or alternatively require chemical labeling with fluorescent tags to achieve measurements at submicrolitre volumes with less sample. Here we present a quantitative and accurate method for the determination of protein stability and the affinity for small molecules, at only 1.5-20 nL optical sample volumes without the need for fluorescent labeling, and that takes advantage of the intrinsic tryptophan fluorescence of most proteins. Coupled to appropriate microfluidic sample preparation methods, the sample requirements could thus be reduced 85,000-fold to just 10 8 molecules. The stability of wild-type FKBP-12 and a destabilizing binding-pocket mutant are studied in the presence and absence of rapamycin, to demonstrate the potential of the technique to both drug screening and protein engineering. The results show that 75% of the interaction energy between FKBP-12 and rapamycin originates from residue Phe99 in the binding site.

show abstract

Section: Introductionmentioning

confidence: 99%

Protein denaturation and protein:drugs interactions from intrinsic protein fluorescence measurements at the nanolitre scale

et al. 2010

View full text Add to dashboard Cite

show abstract

“…In particular, flow separation methods, such as chromatography, 1,2 electrophoresis, [3][4][5][6][7] and field flow fractionation (FFF), [8][9][10] have been extensively used in various fundamental and practical fields, and have facilitated the advancements of modern science. These methods cover separation of almost all classes of materials.…”

mentioning

confidence: 99%

Solute Distribution Coupled with Laminar Flow in Wide-Bore Capillaries: What Can Be Separated without Chemical or Physical Fields?

et al. 2005

View full text Add to dashboard Cite

A number of researches have been devoted to the developments of novel separation principles and methodology, because material separation is one of the most important fundamentals in scientific and technological disciplines. In particular, flow separation methods, such as chromatography, 1,2 electrophoresis, [3][4][5][6][7] and field flow fractionation (FFF), [8][9][10] have been extensively used in various fundamental and practical fields, and have facilitated the advancements of modern science. These methods cover separation of almost all classes of materials. A number of dissolved molecules, from simple ions to macromolecules, can be separated by chromatography or electrophoresis, FFF and electrophoresis being capable of resolving even particles. The dimensions of solutes to be separated by these methods thus range from 10 -10 m to 10 -5 m. This situation may suggest that separation science and technology have been completely matured. However, studying new separation principles is still important, because they can allow the separation based on material properties that have not been utilized for conventional methods, provide higher performance and more efficient separation than existing means, and facilitate the understanding of materials through separation processes.According to chromatographic theories, the intrinsic separation performance of liquid chromatography is restricted in comparison with that of gas chromatography because of much lower solute diffusion in liquid phases. 11However, a liquid flow obviously provides versatile ways for material separation because liquids have higher density and dissolution power than gases, and thus has made possible application to various types of solutes with wide ranges in molecular weights and sizes. In the usual flow separation methods, appropriate chemical (chromatography) or physical (FFF) separation fields are created, which retain solutes to different extents and separate them. A variety of stationary phases for chromatography 2,[12][13][14] and physical forces for FFF [15][16][17][18][19] have been exploited, and have provided different capabilities and selectivity from those performed by conventional separation. In contrast, a separation method that requires neither chemical interactions nor special external fields should also be useful because of its instrumental simplicity. Hydrodynamic chromatography (HDC) is such a case, which allows separation of particles or macromolecules according to their hydrodynamic sizes just by passing them through a narrow channel. [20][21][22][23][24] Particles with larger diameter for example cannot approach the channel wall, and thus flow through the channel faster than the smaller particles, when the laminar flow profile is maintained therein. Although the applicability of this method was very restricted, recent developments of chip technology have brought about a remarkable progress. 23,24 If a capillary of radius a is used for HDC, the time for the elution of a particle of radius rp is given bywhere t0 is the time required ...

show abstract

“…Specific reviews or books dedicated to CE analysis of peptides [4][5][6][7][8][9][10] and proteins [7][8][9][10][11][12][13][14][15] have been published, outlining that the extremely small id of the capillary has the advantage of requiring small sample volumes but often presents poor mass sensitivity. This is observed particularly with UV absorbance detection due to the short optical path length across the capillary.…”

Section: Introductionmentioning

confidence: 99%

LIF detection of peptides and proteins in CE

2007

View full text Add to dashboard Cite

CE- and microchip-based separations coupled with LIF are powerful tools for the separation, detection and determination of biomolecules. CE with certain configurations has the potential to detect a small number of molecules or even a single molecule, thanks to the high spatial coherence of the laser source which permits the excitation of very small sample volumes with high efficiency. This review article discusses the use of LIF detection for the analysis of peptides and proteins in CE. The most common laser sources, basic instrumentation, derivatization modes and set-ups are briefly presented and special attention is paid to the different fluorogenic agents used for pre-, on- and postcapillary derivatization of the functional groups of these compounds. A table summarizing major applications of these derivatization reactions to the analysis of peptides and proteins in CE-LIF and a bibliography with 184 references are provided which covers papers published to the end of 2005.

show abstract

Capillary Electrophoresis for the Analysis of Biopolymers

Cited by 85 publications

References 190 publications

Protein denaturation and protein:drugs interactions from intrinsic protein fluorescence measurements at the nanolitre scale

Protein denaturation and protein:drugs interactions from intrinsic protein fluorescence measurements at the nanolitre scale

Solute Distribution Coupled with Laminar Flow in Wide-Bore Capillaries: What Can Be Separated without Chemical or Physical Fields?

LIF detection of peptides and proteins in CE

Contact Info

Product

Resources

About