Determination of isoelectric points of acidic and basic proteins by capillary electrophoresis

Yao, Yi Ju; Khoo, Kong Soo; Chung, Maxey C. M.; Li, Sam Fong Yau

doi:10.1016/0021-9673(94)85140-9

Cited by 42 publications

(22 citation statements)

References 16 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…1 Knowledge of the value of pI of a protein is particularly useful for separation 2 and purification, 3 and for the characterisation of key physicochemical properties, such as surface charge and solubility, which are typically lower at values of the pH in the vicinity of the pI. 4 A range of conventional methods can be used to determine the isoelectric point of proteins, including isoelectric precipitation, 5 techniques based on ion-exchange adsorption, [6][7][8] zeta potential measurements, [9][10][11] capillary electrophoresis 12 or a recently developed nanoparticle-based approach. 13 However, the predominant way to separate proteins and investigate their pI is isoelectric focusing (IEF), in which a pH gradient is generated across a chamber when an electric field is applied.…”

Section: Introductionmentioning

confidence: 99%

Gradient-free determination of isoelectric points of proteins on chip

Łapińska

Saar

Yates

et al. 2017

Phys. Chem. Chem. Phys.

View full text Add to dashboard Cite

The isoelectric point (pI) of a protein is a key characteristic that influences its overall electrostatic behaviour. The majority of conventional methods for the determination of the isoelectric point of a molecule rely on the use of spatial gradients in pH, although significant practical challenges are associated with such techniques, notably the difficulty in generating a stable and well controlled pH gradient. Here, we introduce a gradient-free approach, exploiting a microfluidic platform which allows us to perform rapid pH change on chip and probe the electrophoretic mobility of species in a controlled field. In particular, in this approach, the pH of the electrolyte solution is modulated in time rather than in space, as in the case for conventional determinations of the isoelectric point. To demonstrate the general approachability of this platform, we have measured the isoelectric points of representative set of seven proteins, bovine serum albumin, β-lactoglobulin, ribonuclease A, ovalbumin, human transferrin, ubiquitin and myoglobin in microlitre sample volumes. The ability to conduct measurements in free solution thus provides the basis for the rapid determination of isoelectric points of proteins under a wide variety of solution conditions and in small volumes.

show abstract

Section: Introductionmentioning

confidence: 99%

Gradient-free determination of isoelectric points of proteins on chip

Łapińska

Saar

Yates

et al. 2017

Phys. Chem. Chem. Phys.

View full text Add to dashboard Cite

show abstract

“…The most ordinarily used wall coatings for modifying the charge on the capillary surface are the following: (nondynamically) covalently bonded/cross-linked polymer [1][2][3], (noncovalently) adsorbed cationic polymers [4][5], (dynamically and noncovalently) adsorbed surfactants [6][7][8], and dynamic double coating (DC) [9][10][11][12]. Nondynamic coating often needs very long coating time.…”

Section: Introductionmentioning

confidence: 99%

A novel double coating for microemulsion electrokinetic chromatography with laser-induced fluorescence detection: as tested with amino acid derivatives

Xie¹,

Chen²,

Zhang³

et al. 2004

Journal of Pharmaceutical and Biomedical Analysis

View full text Add to dashboard Cite

“…The most commonly used wall coatings for modifying the charge on the capillary surface are the following: (nondynamically) covalently bonded/cross-linked polymers [1 -3], (non-covalently) adsorbed cationic polymers [4,5], and (dynamically and non-covalently) adsorbed surfactants [6 -8]. It is known that non-dynamic coatings including conventional trimethylsilyl ones need long coating time, and common dynamic coatings often use polymers or surfactants with complicated molecular structure, which may result in lack of matching with advanced mass spectrometers due to complicated contaminants in the run buffer.…”

Section: Introductionmentioning

confidence: 99%

Micellar electrokinetic chromatography with laser‐induced fluorescence detection for rapid and sensitive analysis of two new bioactive reagents using dynamic covalent coating

Xie

Feng

Chen

et al. 2004

J of Separation Science

View full text Add to dashboard Cite

A simple, rapid, selective, and sensitive micellar electrokinetic chromatography (MEKC) with laser-induced fluorescence detection (LIF) method was developed, using hexamethyldisilazane (HMDS) as dynamic covalent coating (DCC), for the analysis of two new bioactive agents N-n-hexyl-N'-(sodium p-aminobenzenesulfonate) thiourea (HXPT) and N-n-undecyl-N'-(sodium p-aminobenzenesulfonate) thiourea (UPT) derivatized with 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole. MEKC methods both not using DCC and using DCC were investigated. In a series of optimization steps, DCC and a running buffer of 20 mM Na2B4O7 + 16 mM SDS + 8% acetonitrile were applied for determination of the derivatives. Linear relationships for HXPT and UPT were obtained in the range of 5 to 100 microM (correlation coefficient: 0.9986 for HXPT, 0.9978 for UPT), and the detection limits for HXPT and UPT were 16.5 and 39.0 ng mL(-1). The sensitivity was improved over that of fluorescence spectroscopy methods. The method was applied to the analysis of the two reagents in lab water waste with recoveries in the range of 95.6-107.5%.

show abstract

Determination of isoelectric points of acidic and basic proteins by capillary electrophoresis

Cited by 42 publications

References 16 publications

Gradient-free determination of isoelectric points of proteins on chip

Gradient-free determination of isoelectric points of proteins on chip

A novel double coating for microemulsion electrokinetic chromatography with laser-induced fluorescence detection: as tested with amino acid derivatives

Micellar electrokinetic chromatography with laser‐induced fluorescence detection for rapid and sensitive analysis of two new bioactive reagents using dynamic covalent coating

Contact Info

Product

Resources

About