Preparation of intact mitochondria using free-flow isoelectric focusing with post-pH gradient sample injection for morphological, functional and proteomics studies

He, Yu Chen; Kong, Fan Zhi; Fan, Liu Yin; Wu, Jane Y.; Liu, Peter; Li, Jun; Sun, Yan; Zhang, Qiang; Yang, Ying; Wu, Xue Jing; Xiao, Hua; Cao, Cheng-Xi

doi:10.1016/j.aca.2017.06.007

Cited by 15 publications

(15 citation statements)

References 49 publications

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“…For the preparative separation of mitochondria by FF‐IEF He et al. applied a refined technique termed RFFIEF. The system used in this publication is based on the recently developed FFE apparatus containing a gas‐cushion injector and a gravity‐induced self‐balance collector as described above .…”

Section: Purification Of Mitochondriamentioning

confidence: 99%

“…The system used in this publication is based on the recently developed FFE apparatus containing a gas‐cushion injector and a gravity‐induced self‐balance collector as described above . For the separation of mitochondrial subpopulations, a crude mitochondrial preparation from rabbit liver was subjected to the RFFIEF . Compared to a parallel sample injection at the beginning of the IEF, post‐gradient formation sample injection significantly improved resolution of the separation profile giving rise to six organelle fractions differing in their p I .…”

Section: Purification Of Mitochondriamentioning

confidence: 99%

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Preparative free‐flow electrophoresis, a versatile technology complementing gradient centrifugation in the isolation of highly purified cell organelles

Islinger

Wildgruber²,

Völkl

2018

Electrophoresis

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Free-flow electrophoresis (FFE) exploits differences in the overall charge of bio-particles separating cells, organelles, macromolecules, ions, etc. according to their distinct electrophoretic mobility and isoelectric point (pI) values. Indeed, around a neutral pH organelles usually exhibit a negative surface charge, migrating in an electric field from the cathode toward the anode. Since its introduction more than five decades ago by Barrollier et al., Z. Naturforsch. 1958, 13b, 745-755 and Hannig, Z. Anal. Chem. 1961, 181, 244-254, FFE has become an established analytical and preparative separation method for the isolation of a variety of organelles. Particularly, in sophisticated, multistep separating processes to separate subpopulations of organelles, it has gained, meanwhile, a position as a versatile technology and essential element. Relying on the distinct surface charges instead of buoyant densities of cell organelles, the FFE technology is best supporting a preceding centrifugation-based fractionation of subcellular compartments in the second dimension. In the following review, the two-step isolation and purification of subpopulations of classic animal and plant cell organelles will be mainly exemplified.

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Section: Purification Of Mitochondriamentioning

confidence: 99%

Section: Purification Of Mitochondriamentioning

confidence: 99%

Preparative free‐flow electrophoresis, a versatile technology complementing gradient centrifugation in the isolation of highly purified cell organelles

Islinger

Wildgruber²,

Völkl

2018

Electrophoresis

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“…separated complete mitochondria from mitochondria resuspension buffer using the method of free‐flow isoelectric focusing. This method has advantages in measuring the isoelectric point of substances such as proteins [8]. Zeng et al.…”

Section: Introductionmentioning

confidence: 99%

Microalgae separation by inertia‐enhanced pinched flow fractionation

Wang

Liu

et al. 2021

Electrophoresis

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To improve the accuracy and efficiency of ships’ ballast water detection, the separation of microalgae according to size is significant. In this article, a method to separate microalgae based on inertia‐enhanced pinched flow fractionation (iPFF) was reported. The method utilized the inertial lift force induced by flow to separate microalgae according to size continuously. The experimental results show that, as the Reynolds number increases, the separation effect becomes better at first, but then stays unchanged. The best separation effect can be obtained when the Reynolds number is 12.3. In addition, with the increase of the flow rate ratio between sheath fluid and microalgae mixture, the separation effect becomes better and the best separation effect can be obtained when the flow rate ratio reaches 10. In this case, the recovery rate of Tetraselmis sp. is about 90%, and the purity is about 86%; the recovery rate of Chlorella sp. is as high as 99%, and the purity is about 99%. After that, the separation effect keeps getting better but very slowly. In general, this study provides a simple method for the separation of microalgae with different sizes, and lays a foundation for the accurate detection of microalgae in the ballast water.

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“…An electric field is applied perpendicularly to the sample flow to deflect analytes, laterally according to their mobility or p I s, as they continuously flow through the separation channel . Because of its advantages of mild separation environment and solid support medium free characteristics, FFE has been successfully applied to the separation of various biological samples, such as nucleic acids , peptide , proteins , cell organelles , and so on. Moreover, most of the separation modes of CE can be applied to FFE as well , for example, free‐flow zone electrophoresis (FFZE) , free‐flow isoelectric focusing (FFIEF) , and free‐flow isotachophoresis .…”

Section: Introductionmentioning

confidence: 99%

“…In conventional FFIEF, a specific ampholyte buffer solution is required to establish a stable pH gradient . However, preparative FFIEF device requires a large amount of expensive ampholytes, leading to heavy cost.…”

Section: Introductionmentioning

confidence: 99%

Carrier ampholyte‐free free‐flow isoelectric focusing for separation of protein

et al. 2019

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Free‐flow isoelectric focusing (FFIEF) has the merits of mild separation conditions, high recovery and resolution, but suffers from the issues of ampholytes interference and high cost due to expensive carrier ampholytes. In this paper, a home‐made carrier ampholyte‐free FFIEF system was constructed via orientated migration of H+ and OH− provided by electrode solutions. When applying an electric field, a linear pH gradient from pH 4 to 9 (R2 = 0.994) was automatically formed by the electromigration of protons and hydroxyl ions in the separation chamber. The carrier ampholyte‐free FFIEF system not only avoids interference of ampholyte to detection but also guarantees high separation resolution by establishing stable pH gradient. The separation selectivity was conveniently adjusted by controlling operating voltage and optimizing the composition, concentration and flow rate of the carrier buffer. The constructed system was applied to separation of proteins in egg white, followed by MADLI‐TOF‐MS identification. Three major proteins, ovomucoid, ovalbumin and ovotransferrin, were successfully separated according to their pI values with 15 mmol/L Tris‐acetic acid (pH = 6.5) as carrier buffer at a flow rate of 12.9 mL/min.

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Preparation of intact mitochondria using free-flow isoelectric focusing with post-pH gradient sample injection for morphological, functional and proteomics studies

Cited by 15 publications

References 49 publications

Preparative free‐flow electrophoresis, a versatile technology complementing gradient centrifugation in the isolation of highly purified cell organelles

Preparative free‐flow electrophoresis, a versatile technology complementing gradient centrifugation in the isolation of highly purified cell organelles

Microalgae separation by inertia‐enhanced pinched flow fractionation

Carrier ampholyte‐free free‐flow isoelectric focusing for separation of protein

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