Electrophoretic Mobility Distribution Research Articles

New Copper(I) oxide biocatalyst based on functionalized olive stone for the synthesis of 1,4-disubstituted-1,2,3-triazoles under very mild conditions

An efficient methodology for coating silanized olive stone with copper(I) oxide has been described and fully characterized using various techniques, including X-ray photoelectron spectroscopy (XPS), Fourier transform infrared spectroscopy (FTIR-ATR), X-ray fluorescence (XRF), electronic microscopy, thermogravimetric analysis (TGA), moisture content, electrophoretic mobility and particle size distribution. The applicability of this new biocatalyst in the 1,3-dipolar cycloaddition click reaction between alkynes and azides has been demonstrated leading to the obtention of a great variety of 1,4-disubstituted-1,2,3-triazoles with good to excellent conversions. The new biocatalyst showed promising features in terms of recyclability and industrial or biomedical application. It could be recycled up to five times with minimal loss of catalytic activity when synthezing compound 3a-3n. Additionally, it was effective in the multi-gram scale synthesis of compounds 3b and 3e, which were tested for the first time in cells, specifically on the human fibroblast-derived M1 cell line, demonstrating that these compounds are non-cytotoxic and thus suitable for potential uses in biomedicine.

Effect of chemical modification on the distribution of electrophoretic mobilities of individual molecules of E. coli β-galactosidase

Escherichia coli β-galactosidase was labelled with 1 mmol/L fluorescein 5-carbamoylmethylthiopropanoic N-hydroxysuccinimidyl ester for 1 and 3 min. The samples were separated by capillary electrophoresis and peak areas compared to that of labelled BSA for the purpose of quantification of the concentration of attached label. Enzyme concentration in the samples was determined by single molecule counting. The average number of labels attached to each molecule of enzyme was found to be 3.1 and 4.5 when labelled for 1 and 3 min, respectively. The distribution of single enzyme molecule electrophoretic mobilities for the unlabelled enzyme and that labelled for 1 and 3 min were measured using capillary electrophoresis. The average mobilities were found determined to be −(1.99 ± 0.13) × 10–8 m2 V−1 s−1 ( N = 39), −(2.16 ± 0.19) × 10–8 m2 V−1 s−1 ( N = 46), and –(2.18 ± 0.21) × 10–8 m2 V−1 s−1 ( N = 39), respectively. A protein electrophoresis model was applied and predicted that the differences in average mobilities could be explained through relatively minor changes in overall charge, Stokes radius, and shape. This difference was similar to the range in mobilities observed in the unlabelled protein. This is consistent with the electrophoretic heterogeneity of the unmodified enzyme being caused by relatively small differences in charge, size, and shape of the individual molecules in the population.

Using Capillary Electrophoresis to Investigate Protein Conformational and Compositional Heterogeneity.

Detailed insights into protein structure/function relationships require robust characterization methodologies. Free-solution capillary electrophoresis (CE) is a unique separation technique which is sensitive to the conformation and/or composition of proteins, and therefore provides information on the heterogeneity of these properties. Three unrelated, conformationally/compositionally-altered proteins were separated by CE. An electrophoretic mobility distribution was determined for each protein along with its conformational and/or compositional heterogeneity. The CE results were compared with molar mass distributions obtained from size-exclusion chromatography coupled to light scattering (SEC-MALS). Bovine serum albumin multimers and two monomeric species were separated, highlighting variations in conformational/compositional heterogeneity among the multimers. Analysis of yeast alcohol dehydrogenase resolved two monomeric conformers and various tetrameric species, illustrating the impact of zinc ion removal and disulfide bond reduction on the protein's heterogeneity. The apo (calcium-free) and holo forms of bovine α-lactalbumin were separated and differences in the species' heterogeneity were measured; by contrast, the SEC-MALS profiles were identical. Comparative analysis of these structurally unrelated proteins provided novel insights into the interplay between molar mass and conformational/compositional heterogeneity. Overall, this study expands the utility of CE by demonstrating its capacity to discern protein species and their heterogeneity, properties which are not readily accessible by other analytical techniques.

Clay–Magnetite Co-Aggregates for Efficient Magnetic Removal of Organic and Inorganic Pollutants

This work reports the behavior of montmorillonite–magnetite mixtures of varying composition in aqueous dispersions and evaluates their adsorbing properties using a cationic organic pollutant, methylene blue (MB+), and an anionic inorganic pollutant, arsenate (As(V)), as the adsorbing species. The effects of the presence of montmorillonite on the As(V) adsorption by magnetite and the effects of magnetite on the MB+ adsorption by the clay were specially addressed. The simple mixture of a montmorillonite dispersion with a magnetite dispersion led to the spontaneous formation of montmorillonite–magnetite co-aggregates. These co-aggregates showed a unimodal electrophoretic mobility distribution, with no evidence of the presence of separate populations of montmorillonite or magnetite. The application of a magnetic field confirmed the formation of co-aggregates and showed that their separation rate increased as the magnetite content increased. Adsorption studies as a function of the aggregate composition demonstrated that MB+ uptake was mainly controlled by the content of montmorillonite, while As(V) adsorption was mainly controlled by the content of Fe3O4. This permits an easy tuning of the adsorbing properties of cations and anions by controlling the composition of the system.

Characterization and differential retention of Q beta bacteriophage virus-like particles using cyclical electrical field-flow fractionation and asymmetrical flow field-flow fractionation.

Virus-like particles (VLPs) are widely used in medicine, but can be difficult to characterize and isolate from aggregates. In this research, primarily cyclical electrical field-flow fractionation (CyElFFF) coupled with multi-angle light scattering (MALS), and dynamic light scattering (DLS) detectors, was used for the first time to perform size and electrical characterization of three different types of Q beta bacteriophage virus-like particles (VLPs): a blank Q beta bacteriophage which is denoted as VLP and two conjugated ones with different peptides. The CyElFFF results were verified with transmission electron microscopy (TEM). Asymmetrical flow field-flow fractionation (AF4) coupled with MALS was also applied using conditions similar to those used in the CyElFFF experiments, and the results of the two techniques were compared to each other. Using these techniques, the size and electrophoretic characteristics of the fractionated VLPs in CyElFFF were obtained. The results indicate that CyElFFF can be used to obtain a clear distribution of electrophoretic mobilities for each type of VLP. Accordingly, CyElFFF was able to differentially retain and isolate VLPs with high surface electric charge/electrophoretic mobility from the ones with low electric charge/electrophoretic mobility. Regarding the size characterization, the size distribution of the eluted VLPs was obtained using both techniques. CyElFFF was able to identify subpopulations that did not appear in the AF4 results by generating a shoulder peak, whereas AF4 produced a single peak. Different size characteristics of the VLPs appearing in the shoulder peak and the main peak indicate that CyElFFF was able to isolate aggregated VLPs from the monomers partially. Graphical abstract.

Characterization of Diblock Copolymers by Capillary Electrophoresis: From Electrophoretic Mobility Distribution to Distribution of Composition

Free solution capillary-electrophoresis (CE) is a powerful separation technique for the characterization of diblock copolymers. In this work, four series of double-hydrophilic anionic and cationic block copolymers, namely, poly(acrylamide)-block-poly(acrylic acid) (PAM-b-PAA), poly(acrylamide)-block-poly((3-acrylamidopropyl)trimethylammonium chloride) (PAM-b-PAPTAC), poly(ethylene oxide)-block-poly(acrylic acid) (PEO-b-PAA) and poly(poly(ethylene glycol) methyl ether acrylate)-block-poly(acrylic acid) (P(PEGA)-b-PAA), were synthesized by reversible additionfragmentation chain transfer (RAFT) polymerization and characterized by CE. The electrophoretic mobility distributions of the copolymers were transformed into distributions of composition ratio by introducing a retardation parameter, Xexp,, that represents the hydrodynamic drag retardation due to the neutral block of the copolymer. A linear correlation between Xexp and the ratio of the degrees of polymerization of each blocks was experimentally established and was consistent with the model of electrophoretic mobility of composite macromolecules with hydrodynamic coupling. Finally, the comparison of the distributions between the different copolymer families was significantly improved by considering the distributions in composition ratio compared to the electrophoretic mobility distributions, since it takes into account the differences in solvation, expansion and drag force according to the chemical nature of the blocks.

Correlation of electrophoretic mobility with exfoliation of montmorillonite platelets in aqueous solutions

In this work, the effect of the exfoliation degree of montmorillonite (MMT) platelets on their electrophoretic mobility distributions in aqueous suspensions was investigated in order to approach into the correlation of MMT exfoliation with electrokinetic property. The experimental results have shown that the negative mobility increased with the increase of exfoliation degree or with the decrease of MMT thickness. This observation might be attributed to the “exposing” of permanent layer negative charge during the exfoliation processing and the “spillover” of electrostatic potential from basal surfaces onto edges. The former would increase the permanent negative charge of the MMT platelets because of the large amounts of layer charge; the latter would decrease the positive edge charge. It is demonstrated that MMT exfoliation would change the electrokinetic property, which might be helpful of the applications of MMT in the related industries.

Effect of transfer agent, temperature and initial monomer concentration on branching in poly(acrylic acid): A study by 13C NMR spectroscopy and capillary electrophoresis

Branching has been investigated in poly(acrylic acid) synthesized by conventional radical polymerization with and without chain transfer agent (CTA) at different temperatures and initial monomer concentrations. The average number of branches per monomer unit (i.e. degree of branching) was quantified by solution-state 13C NMR spectroscopy. The heterogeneity of branching (dispersity of the electrophoretic mobility distributions) was measured by capillary electrophoresis in the critical conditions (CE-CC). The degree of branching (DB) increases with the reaction temperature due to a rise in the frequency of reactions leading to branches, while the heterogeneity of branching remains steady. DB is lower in polymer synthesized with CTA. This decrease is due to either the CTA quenching the mid-chain radicals or a reduction of the rate of chain transfer to polymer relative to (chain-end) propagation. No influence of initial monomer concentration on DB and on the heterogeneity of branching was observed.

Electrophoretic mobility study for heterocoagulation of montmorillonite with fluorite in aqueous solutions

In this work, the heterocoagulation of fine montmorillonite (MMT) and fluorite particles in aqueous suspensions was investigated through the measurement of electrophoretic mobility distributions. The experimental results have shown that the heterocoagulation closely correlated to the electrophoretic mobility distribution of the mixed mineral dispersion. If a strong heterocoagulation happened, a single-modal electrophoretic mobility distribution with only one peak could be determined, and the peak located between the peaks of the two individual minerals. Without heterocoagulation, a bimodal electrophoretic mobility distribution with two peaks could be observed, which was consistent with the peaks of the two individual minerals. It is demonstrated that the heterocoagulation of two mineral fines in aqueous suspensions could be predicted by the electrophoretic mobility distribution of the mixed mineral dispersion.

Determination of the distributions of degrees of acetylation of chitosan

Chitosan is often characterized by its average degree of acetylation. To increase chitosan’s use in various industries, a more thorough characterization is necessary as the acetylation of chitosan affects properties such as dissolution and mechanical properties of chitosan films. Despite the poor solubility of chitosan, free solution capillary electrophoresis (CE) allows a robust separation of chitosan by the degree of acetylation. The distribution of degrees of acetylation of various chitosan samples was characterized through their distributions of electrophoretic mobilities. These distributions can be obtained easily and with high precision. The heterogeneity of the chitosan chains in terms of acetylation was characterized through the dispersity of the electrophoretic mobility distributions obtained. The relationship between the number-average degree of acetylation obtained by solid-state NMR spectroscopy and the weight-average electrophoretic mobilities was established. The distribution of degrees of acetylation was determined using capillary electrophoresis in the critical conditions (CE-CC).

Overcharging of magnetite nanoparticles in electrolyte solutions

The effect of electrolytes with mono-, bi-, and trivalent anions on the surface charge (ζ-potential) of magnetite nanoparticles was investigated. The nanoparticles of magnetite, Fe3O4, were synthesised using different methods: a) coprecipitation of bivalent and trivalent iron sulphates at temperature T=25°C (sample NM1) and b) oxidation of rotating steel disk in distilled water at T=25°C (sample NM2) and at T=50°C (sample NM3). All samples were characterized using transmission electron microscopy, infra-red spectroscopy, by measurements of electrophoretic mobility and particle size distribution. Transmission electron microscopy data evidenced that primary particles in dried powder samples have quasi-spherical shape with mean diameters of ⿿15nm (Nп1), ⿿80nm (Nп2) and ⿿70nm (Nп3). From other hand data of particle size distribution analysis in aqueous suspensions at pH 6 indicated formation of aggregates of size of ⿿30nm (Nп1), ⿿1200nm (Nп2) and ⿿520nm (Nп3). Infra-red data also evidenced the presence of different functional groups in dependence on the protocol of synthesis. In presence of background electrolyte KCl (1 mM) the isoelectric point for all particles was in the interval ѿп 6.0⿿8.0. Adding of electrolytes with mono- (Cl⿿), bi- (SO42⿿), and trivalent (PO43⿿) anions resulted in noticeable differences in behaviour of the ζ-potential and position of the isoelectric point for particles Nп1, Nп2 and Nп3. An increase of concentration of electrolytes always resulted in overcharging (changing from positive to negative charge) of particles Nп1 and NM3, whereas for particle Nп2 the overcharging was observed for both bivalent (SO42⿿) and trivalent (PO43⿿) anions. This behaviour cannot be explained by changes in the structure of electric double layer. The observed shifts of the isoelectric point and changes in the pH values in the presence of electrolytes reflected the impact of specific adsorption of anions on the ζ-potential of magnetite nanoparticles. Differences in pH values at which overcharging of nanoparticles took place can be explained by diverse structure of the surfaces of particles Nп1, Nп2 and Nп3.

Conformational change in individual enzyme molecules.

Single β-galactosidase molecule assays were performed using a capillary electrophoresis based protocol, employing post-column laser-induced fluorescence detection. In a first set of experiments, the distribution of single β-galactosidase molecule catalytic rates and electrophoretic mobilities were determined from lysates of Escherichia coli strains containing deletions for different heat shock proteins and grown under normal and heat shock conditions. There was no clear observed pattern of effect of heat shock protein expression on these distributions. In a second set of experiments, individual enzyme molecule catalytic rates were determined at 21 °C before and after 2 sequential brief periods of incubation at 50, 28, and 10 °C. The brief incubations at 50 °C caused a change in the enzyme molecules resulting in a different catalytic rate. Any given molecule was just as likely to show an increase in rate as a decrease, resulting in no significant difference in the average rate of the population. The average change in individual molecule rate was dependent upon the temperature of the brief incubation period, with a lesser average change occurring at 28 °C and negligible change at 10 °C. A third set of experiments was similar to that of the second with the exception that it was electrophoretic mobility that was considered. This provided a similar result. Incubation at higher temperature resulted in a change in electrophoretic mobility. The probability of an individual molecules switching to a higher mobility was approximately equal to that of switching to a lower mobility, resulting in no net average change in the population. The magnitude of the changes in electrophoretic mobilities suggest that the associated conformational changes are subtle.

Characterization of gold nanoparticles with different hydrophilic coatings via capillary electrophoresis and Taylor dispersion analysis. Part I: Determination of the zeta potential employing a modified analytic approximation

Taking gold nanoparticles with different hydrophilic coatings as an example, it is investigated whether capillary electrophoresis in combination with Taylor dispersion analysis allows for the precise determination of mean electrophoretic mobilities, electrophoretic mobility distributions, and zeta potentials in a matrix of exactly known composition and the calibration-free determination of number-weighted mean hydrodynamic radii. Our experimental data confirm that the calculation of the zeta potential for colloidal nanoparticles with ζ>25mV requires to take the relaxation effect into account. Because of the requirement to avoid particle–wall interactions, a solution of disodiumtetraborate decahydrate (borax) in deionized water had been selected as suitable electrolyte. Measurements of the electrophoretic mobility at different ionic strength and application of the analytic approximation developed by Ohshima show that in the present case of a buffered solution with a weak electrolyte co-ion and a strong electrolyte counterion, the effective ionic drag coefficient should be approximated with the ionic drag coefficient of the counterion. The obtained results are in good agreement with theoretical expectations regarding the dependence of the zeta potential and the electrokinetic surface charge density on the ionic strength. We also show that Taylor dispersion analysis (besides estimation of the number-weighted mean hydrodynamic radius) provides additional information on the type and width of the number-weighted particle distribution.

Nanoparticle size distributions measured by optical adaptive-deconvolution passivated-gel electrophoresis

We image visible light scattered from dispersions of charged spherical nanoparticles propagating through a passivated agarose gel during electrophoresis. By analyzing one-dimensional light intensities along different lanes, we measure the mobility distributions of the nanoparticles and thereby infer their size distributions, which become time-independent after adequate propagation and separation have occurred. For a given large-pore passivated agarose gel, experiments using monodisperse, surfactant-free, sulfate-stabilized, polystyrene nanopheres establish the propagation distance as a function of time for a range of different sphere radii having known surface charges. As bands of monodisperse nanospheres propagate through the gel, the bands become smeared, developing asymmetric tails as some nanospheres experience additional delays compared to others of the same size. After background subtraction, these bands, including their tails, can be fit well using a modified log–normal distribution, yielding deconvolution parameters that vary with propagation distance and transit time. To demonstrate the approach for complex nanosphere dispersions, such as a multi-modal mixture or a broadly polydisperse nanoemulsion, we measure scattered light intensities as a function of propagation distance and time during gel-EP. Iterative deconvolution using a modified log–normal point-spread function, which changes shape according to propagation distance and time, directly yields unsmeared, high-resolution electrophoretic mobility distributions, from which detailed particle size distributions are inferred.

Simultaneous Measurement of Individual Mitochondrial Membrane Potential and Electrophoretic Mobility by Capillary Electrophoresis

Mitochondrial membrane potential varies, depending on energy demand, subcellular location, and morphology and is commonly used as an indicator of mitochondrial functional status. Electrophoretic mobility is a heterogeneous surface property reflective of mitochondrial surface composition and morphology, which could be used as a basis for separation of mitochondrial subpopulations. Since these properties are heterogeneous, methods for their characterization in individual mitochondria are needed to better design and understand electrophoretic separations of subpopulations of mitochondria. Here we report on the first method for simultaneous determination of individual mitochondrial membrane potential and electrophoretic mobility by capillary electrophoresis with laser-induced fluorescence detection (CE-LIF). Mitochondria were isolated from cultured cells, mouse muscle, or liver, and then polarized, labeled with JC-1 (a ratiometric fluorescent probe, which indicates changes in membrane potential), and separated with CE-LIF. Red/green fluorescence intensity ratios from individual mitochondria were used as an indicator of mitochondrial membrane potential. Reproducible distributions of individual mitochondrial membrane potential and electrophoretic mobility were observed. Analysis of polarized and depolarized regions of interest defined using red/green ratios and runs of depolarized controls allowed for the determination of membrane potential and comparison of electrophoretic mobility distributions in preparations containing depolarized mitochondria. Through comparison of these regions of interest, we observed dependence of electrophoretic mobility on membrane potential, with polarized regions of interest displaying decreased electrophoretic mobility. This method could be applied to investigate mitochondrial heterogeneity in aging or disease models where membrane potential is an important factor.

Describing autophagy via analysis of individual organelles by capillary electrophoresis with laser induced fluorescence detection.

Autophagy is a cellular process responsible for the degradation of intracellular cargo. Its dynamic nature and the multiple types of autophagy organelles present at a given time make current measurements, such as those done by Western blotting, insufficient to understand autophagy and its roles in aging and disease. Capillary electrophoresis coupled to laser induced fluorescence detection (CE-LIF) has been used previously to count and determine properties of individual organelles, but has never been used on autophagy organelles or for determination of changes of such properties. Here we used autophagy organelles isolated from L6 cells expressing GFP-LC3, which is an autophagy marker, to develop a CE-LIF method for the determination of the number of autophagy organelles, their individual GFP-LC3 fluorescence intensities, and their individual electrophoretic mobilities. These properties were compared under basal and rapamycin-driven autophagy, which showed differences in the number of detected organelles and electrophoretic mobility distributions of autophagy organelles. Vinblastine treatment was also used to halt autophagy and further characterize changes and provide additional insight on the nature of autophagy organelles. This approach revealed dramatic and opposite directions in changes of organelle numbers, GFP-LC3 contents, and electrophoretic mobilities during the duration of the vinblastine treatment. These trends suggested the identity of organelle types being detected. These observations demonstrate that individual organelle analysis by CE-LIF is a powerful technology to investigate the complexity and nature of autophagy, a process that plays critical roles in response to drug treatments, aging, and disease.

High Frequency Spontaneous Deletions within the IcaADBC Operon of Clinical Staphylococcus epidermidis Isolates.

Staphylococcus epidermidis has been shown to undergo a phase variation correlating with expression of the icaADBC operon which contributes to biofilm formation. Biofilm formation of Enterococcus faecalis is related to heterogeneity in electrophoretic mobility. Here the relationship between phase variants of clinical isolates of S. epidermidis, icaADBC presence and electrophoretic mobility distributions is investigated. Of 105 S. epidermidis clinical isolates, 5 showed phase variation on Congo Red agar plate. Biofilm forming capability of the black colonies and inability of the red colonies were confirmed using a microtiter plate assay and confocal laser scanning microscopy. Upon analysis of electrophoretic mobility distributions, the black colonies displayed heterogeneity at pH 2 which was absent in the red colonies of the same strain. Surprisingly, it was shown that in all red colonies had lost the icaADBC genes. Determination of gene copy number using Real Time PCR targeting icaA showed reduction of gene copy within a culture with phase variation. In conclusion, using three fundamentally different approaches phase variation of the five clinical isolates was observed. Variants appeared through loss of icaA and icaC gens. To our knowledge this is the first report indicating S. epidermidis strains irreversible switching from biofilm + to biofilm – phenotype by deletion of ica genes. Key words: deletion, ica genes, Staphylococcus epidermidis, IcaADBC operon

Characterization of Polymerized Liposomes Using a Combination of dc and Cyclical Electrical Field-Flow Fractionation

Characterization of polymerized liposomes (PolyPIPosomes) was carried out using a combination of normal dc electrical field-flow fractionation and cyclical electrical field-flow fractionation (CyElFFF) as an analytical technique. The constant nature of the carrier fluid and channel configuration for this technique eliminates many variables associated with multidimensional analysis. CyElFFF uses an oscillating field to induce separation and is performed in the same channel as standard dc electrical field-flow fractionation separation. Theory and experimental methods to characterize nanoparticles in terms of their sizes and electrophoretic mobilities are discussed in this paper. Polystyrene nanoparticles are used for system calibration and characterization of the separation performance, whereas polymerized liposomes are used to demonstrate the applicability of the system to biomedical samples. This paper is also the first to report separation and a higher effective field when CyElFFF is operated at very low applied voltages. The technique is shown to have the ability to quantify both particle size and electrophoretic mobility distributions for colloidal polystyrene nanoparticles and PolyPIPosomes.

Characterization of Carboxylated Nanolatexes by Capillary Electrophoresis

Poly(styrene-co-acrylic acid) (St/AA) and poly(styrene-co-methacrylic acid) (St/MA) nanolatexes with different acid contents were prepared by emulsion copolymerization and were analyzed by capillary electrophoresis (CE) and by laser doppler velocimetry (LDV). Due to the intrinsic differences in the methodologies, CE (separative technique) and LDV (zetametry, nonseparative technique) lead to very different electrophoretic mobility distributions. Beyond these differences, the variation of the electrophoretic mobility is a complex and nonlinear function of the hydrodynamic radius, the ionic strength, and the zeta potential. To gain better insight on the influence of the ionic strength and the acid content on the electrophoretic behavior of the nanolatexes, the electrophoretic mobility data were changed into surface charge densities using the O'Brien, White, and Ohshima modeling. This approach leads to the conclusion that the surface charge density is mainly controlled at high ionic strength (∼50 mM) by the adsorption of anionic surfactants coming from the sample. On the contrary, at low ionic strength, and/or in the presence of neutral surfactant in the electrolyte, the acid content was the main parameter controlling the surface charge density of the nanolatexes.

Capillary electrophoretic analysis reveals subcellular binding between individual mitochondria and cytoskeleton.

Interactions between the cytoskeleton and mitochondria are essential for normal cellular function. An assessment of such interactions is commonly based on bulk analysis of mitochondrial and cytoskeletal markers present in a given sample, which assumes complete binding between these two organelle types. Such measurements are biased because they rarely account for nonbound "free" subcellular species. Here we report on the use of capillary electrophoresis with dual laser induced fluorescence detection (CE-LIF) to identify, classify, count, and quantify properties of individual binding events of the mitochondria and cytoskeleton. Mitochondria were fluorescently labeled with DsRed2 while F-actin, a major cytoskeletal component, was fluorescently labeled with Alexa488-phalloidin. In a typical subcellular fraction of L6 myoblasts, 79% of mitochondrial events did not have detectable levels of F-actin, while the rest had on average ~2 zmol of F-actin, which theoretically represents a ~2.5 μm long network of actin filaments per event. Trypsin treatment of L6 subcellular fractions prior to analysis decreased the fraction of mitochondrial events with detectable levels of F-actin, which is expected from digestion of cytoskeletal proteins on the surface of mitochondria. The electrophoretic mobility distributions of the individual events were also used to further distinguish between cytoskeleton-bound from cytoskeleton-free mitochondrial events. The CE-LIF approach described here could be further developed to explore cytoskeleton interactions with other subcellular structures, the effects of cytoskeleton destabilizing drugs, and the progression of viral infections.