Study on stability mechanism of immobilized pH gradient in isoelectric focusing via the Svensson–Tiselius differential equation and moving reaction boundary

Guo, Chaowan; Li, Si; Wang, Hou-Yu; Zhang, Dong; Li, Guoqing; Zhang, Jie; Fan, Liu-Yin; Cao, Cheng-Xi

doi:10.1016/j.talanta.2013.03.026

Cited by 12 publications

(7 citation statements)

References 31 publications

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“…In Panel B, the RSD values of p I 6.8, 7.04, 7.09, and 7.13 for the samples of Group 2 were respectively 4.79, 5.19, 3.66, and 2.10%, and in Panel C the ones of p I 6.8, 7.04, 7.09, and 7.13 for the samples of Group 3 were 6.27, 3.92, 2.17, and 2.77%, respectively. All the data suggested fair stability and reliability of the protocol due to the IPG technique and equivalent fluxes of hydrogen and hydroxyl ions .…”

Section: Resultsmentioning

confidence: 83%

“…However, if the grind time was more than 30 min, long focusing time and high current were observed in the IEF run, resulting in a poor electrofocusing (). This was due to the factor that more salt was released into the extracting sample from the meat tissue if long‐term extraction was given, leading to poor IEF results .…”

Section: Resultsmentioning

confidence: 99%

“…IPG strips (7 cm; pH 3–10), 40% v/v CA (pH 3–10) and 50% v/v glycerol were used for the separation of Hb and Mb extracted from the chicken muscle sample. Rehydration of IPG strips was carried out in accordance with the manufacturer's instruction . The rehydration buffer (total 125 µL) for each IPG gel strip contained 25 µL glycerol (20%, v/v), 1.9 µL 40% CA (0.5%, v/v), 300 µg protein sample, and 50–100 µL ultrapure water.…”

Section: Methodsmentioning

confidence: 99%

See 2 more Smart Citations

A facile isoelectric focusing of myoglobin and hemoglobin used as markers for screening of chicken meat quality in China

Saud

Sun

et al. 2019

Electrophoresis

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A novel analytical protocol was developed for general quality screening of chicken meat based on IEF and protein extraction. To demonstrate the developed protocol, 24 chickens were divided into three groups; each had eight chickens. The chickens in Group 1 were slaughtered by exsanguination, Group 2 asphyxiated in water, and that in Group 3 were infected by new castle disease virus. Proteins were extracted from the meat samples by using pure water as an extractant, separated by IEF, verified by western blot, and quantified via imaging analysis. The relevant experiments demonstrated that two myoglobin (Mb) bands were detected at pI 6.8 and 7.04 for all samples of Groups 1, 2, and 3, but there were additional hemoglobin (Hb) bands at pI 7.09 and 7.13 (P < 0.05) for the samples of Groups 2 and 3. The results implied that Hb bands might be a potential biomarker for the screening of chicken meat quality. The RSD values of two Mb bands (pI 6.8 and 7.04) in Group 1 were respectively 4.08 and 3.63%, the ones of two Hb bands (pI 7.09 and 7.13) in Group 2 were 3.66 and 2.10%, and those in Group 3 were 2.17% and 2.77%, respectively. All the RSD values indicated high stability and reliability of the developed protocol. Additionally, the protocol had a direct readout of protein bands in IEF without staining. However, it was time‐consuming and had high cost. Even so, the relevant general method and finding have potential for screening of chicken meat quality.

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Section: Resultsmentioning

confidence: 83%

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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A facile isoelectric focusing of myoglobin and hemoglobin used as markers for screening of chicken meat quality in China

Saud

Sun

et al. 2019

Electrophoresis

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“…It must be also noted that the mathematical model for stream broadening described in this manuscript is only applicable to systems with a stable pH gradient. In assays where this gradient fluctuates temporally due to uncontrollable factors [41,42] or is intentionally varied with time [43,44], the noted analysis may not be valid. In addition, it has been recently suggested that the production of water in isoelectric focusing system can also lead to zone broadening which again has not been accounted for in the current study [41,45].…”

Section: Resultsmentioning

confidence: 99%

Broadening of analyte streams due to a transverse pressure gradient in free-flow isoelectric focusing

Dutta

2017

Journal of Chromatography A

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Pressure-driven cross-flows can arise in free-flow isoelectric focusing systems (FFIEF) due to a non-uniform electroosmotic flow velocity along the channel width induced by the pH gradient in this direction. In addition, variations in the channel cross-section as well as unwanted differences in hydrostatic heads at the buffer/sample inlet ports can also lead to such pressure-gradients which besides altering the equilibrium position of the sample zones have a tendency to substantially broaden their widths deteriorating the separations. In this situation, a thorough assessment of stream broadening due to transverse pressure-gradients in FFIEF devices is necessary in order to establish accurate design rules for the assay. The present article describes a mathematical framework to estimate the noted zone dispersion in FFIEF separations based on the method-of-moments approach under laminar flow conditions. A closed-form expression has been derived for the spatial variance of the analyte streams at their equilibrium positions as a function of the various operating parameters governing the assay performance. This expression predicts the normalized stream variance under the chosen conditions to be determined by two dimensionless Péclet numbers evaluated based on the transverse pressure-driven and electrophoretic solute velocities in the separation chamber, respectively. Moreover, the analysis shows that while the stream width can be expected to increase with an increase in the value of the first Péclet number, the opposite trend will be followed with respect to the latter. The noted results have been validated using Monte Carlo simulations that also establish a time/length scale over which the predicted equilibrium stream width is attained in the system.

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“…Moreover, the solubilizing agents must be compatible with isoelectric focusing (IEF) and preserve the protein solubilization at critical steps, e.g., at the sample entry into the gel and/or when protein achieves its isoelectric point [29]. Nevertheless, other strategies should be considered to avoid gel-to-gel variation, including the refinement and tuning of protein separation by isoelectric point (pI) [30][31][32][33][34] and by molecular weight (MW) [33,35,36].…”

Section: Introductionmentioning

confidence: 99%

Refinement of two-dimensional electrophoresis for vitreous proteome profiling using an artificial neural network

et al. 2019

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Despite technological advances, two-dimensional electrophoresis (2DE) of biological fluids, such as vitreous, remains a major challenge. In this study, artificial neural network was applied to optimize the recovery of vitreous proteins and its detection by 2DE analysis through the combination of several solubilizing agents (CHAPS, Genapol, DTT, IPG buffer), temperature, and total voltage. The highest protein recovery (94.9% ± 4.5) was achieved using 4% (w/v) CHAPS, 0.1% (v/v) Genapol, 20 mM DTT, and 2% (v/v) IPG buffer. Two iterations were required to achieve an optimized response (580 spots) using 4% (w/v) CHAPS, 0.2% (v/v) Genapol, 60 mM DTT, and 0.5% (v/v) IPG buffer at 35 kVh and 25 °C, representing a 2.4-fold improvement over the standard initial conditions of the experimental design. The analysis of depleted vitreous using the optimized protocol resulted in an additional 1.3-fold increment in protein detection over the optimal output, with an average of 761 spots detected in vitreous from different vitreoretinopathies. Our results clearly indicate the importance of combining the appropriate amount of solubilizing agents with a suitable control of the temperature and voltage to obtain high-quality gels. The high-throughput of this model provides an effective starting point for the optimization of 2DE protocols. This experimental design can be adapted to other types of matrices.

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Study on stability mechanism of immobilized pH gradient in isoelectric focusing via the Svensson–Tiselius differential equation and moving reaction boundary

Cited by 12 publications

References 31 publications

A facile isoelectric focusing of myoglobin and hemoglobin used as markers for screening of chicken meat quality in China

A facile isoelectric focusing of myoglobin and hemoglobin used as markers for screening of chicken meat quality in China

Broadening of analyte streams due to a transverse pressure gradient in free-flow isoelectric focusing

Refinement of two-dimensional electrophoresis for vitreous proteome profiling using an artificial neural network

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