The Application of Electrophoresis in Soil Research

Abstract

Soil is a complex mixture of minerals and organic matters in which microbes, plants, and animals interact. In the natural environment, soil constantly undergoes physical, chemical, and biological transformations under the influences of environmental factors such as humidity and temperature. Studies on soil chemical compositions, microbes, and abundances of plants and animals provide useful information on the soil property for proper land use planning. Since soil is a complex mixture, soil studies require the effective separation of its various components, which can be achieved with electrophoresis, a powerful method that exploits the inherent differences in the physical and chemical properties of these components. By combining electrophoresis with other technologies such as chromatography, mass spectrometry, polymerase chain reaction (PCR), and DNA sequencing, substances including humic acids, amino acids, environmental pollutants, nutrients, and microbial, plant, and animal DNA can be identified and quantified. In this chapter, the applications of different electrophoresis-based technologies will be discussed with respect to soil research, and their principles, advantages, and limitations will be addressed.

1. Introduction

The soil is a complex ecosystem in which inorganic and organic compounds, microbes, plants, and animals co-exist. The composition of soil is dynamic since it is heavily dependent on environmental conditions such as humidity and temperature. In the face of a changing climate, the soil composition fluctuates even more. Soil quality provides important information for the proper planning of land use, and is greatly influenced by microbial activities. For example, the degradation of soil organic matter (SOM) and the nitrogen-fixing and/or denitrifying activities of soil-borne microbes regulate the fluxes of carbon and nitrogen [1], which is important for the growth of all organisms in soil.

Electrophoresis is a versatile technique for studying the chemical and microbial compositions of soil, separating molecules based on their sizes and charges. To improve the resolution, variants of electrophoresis such as capillary electrophoresis, denaturing electrophoresis, and two-dimensional electrophoresis were developed. To further enhance the analytical capacity of electrophoresis, the technique has been coupled with other platforms, such as mass spectrometry and DNA sequencing, to reveal the identity of the molecules. In addition to resolving proteins and DNA, electrophoresis is also employed to prepare DNA extracted from soil for downstream analyses. The study of soil environmental DNA (eDNA) provides information on the identities and abundances of plants and animals in soil. Thus, electrophoresis has been demonstrated as a useful tool for soil research.

2. The application of capillary electrophoresis (CE) in soil chemical composition analyses

The chemical composition of soil includes soluble substances such as humic acids, amino acids, peptides, proteins, oligonucleotides, carbohydrates, pigments, toxins, pesticides, vitamins, and chiral compounds [2]. Capillary electrophoresis (CE) has been proven to be a powerful analytical technique for separating compounds within a sample [3]. The method was originally developed using glass tubes to separate solutes [4]. The advantage of CE is that it requires only a small amount of sample to produce a high-resolution output in a short amount of time. Due to its efficiency in separating different compounds, CE is widely used in forensic studies [5], biomedical sciences [6], and soil research [7].

2.1 General principle of CE

CE features the use of a capillary tube, usually with an inner diameter ranging from 20 to 200 μm, for analyte separation [8]. The small-diameter capillary enhances the resolution by reducing lateral sample diffusion and minimizing the temperature difference between the center and the wall of the capillary [8]. CE encompasses a diverse range of techniques that serve distinct analytical purposes. For instance, capillary zone electrophoresis separates analytes based on their charge-to-size ratios, while capillary isoelectric focusing (CIEF) separates analytes by exploiting their different isoelectric points. The experimental setup for each technique is tailored to suit its specific requirements. In the case of capillary zone electrophoresis, a fused silica capillary is employed to connect two electrolyte buffers, which are in turn connected to opposing electrodes [9]. Upon the application of voltage, the sample flows through the capillary and its components are separated based on their charge-to-size ratios [9]. Detection and identification of the separated components are achieved using techniques such as UV-absorbance, fluorescence, and mass spectrometry [10].

2.2 Applications of different CE techniques

2.2.1 Capillary zone electrophoresis for humic acid and amino acid analyses

In soil, the decomposition of plant and animal matters containing oxygen-containing functional groups, such as ketones and carboxyl groups, results in humic substances (HSs) [11]. The humic fingerprint of a soil provides information on the soil property, palaeo-history, and pedogenesis [12]. The humic fingerprints of different layers of soil in a particular site is also useful for reconstructing the site history [12]. CE is a powerful technique for HS separation based on their charge-to-size ratios. For example, capillary zone electrophoresis could be used to generate the humic acid fingerprints of different soils [13]. The analyses revealed that humic acids from different soils exhibited different migration patterns and UV absorption profiles, allowing for their differentiation.

Amino acids serve as the building blocks of proteins and perform a variety of critical biological functions. In soil, amino acids play an important role in the nitrogen cycle in ecosystems. CE coupled with laser-induced fluorescence (CE-LIF) is a powerful tool for the detection of amino acids in soil. This technique involves the excitation of amino acids with laser light to a higher energy level, followed by the detection of the different emission wavelengths from different amino acids [14]. Using this approach, 17 common amino acids can be effectively separated and detected in 12 minutes [15].

Traditionally, chromatographic techniques have been widely used to analyze amino acids in soil [16]. Compared to the traditional techniques, CE offers several advantages including simpler procedures and greater accuracy. In contrast to chromatographic techniques, the CE-LIF analysis of amino acids in soil samples does not require a desalting step to remove any contamination from inorganic ions [16]. CE also provides a better resolution in amino acid separation in soil sample analyses [17]. However, compared to certain chromatographic techniques such as HPLC, the maximum amount of sample that can be applied to CE is much lower. Such a low input limits the detection capability of CE, but this limitation can be mitigated by the combined use of LIF [15].

2.2.2 Capillary isoelectric focusing for humic acid analysis

Besides capillary zone electrophoresis, capillary isoelectric focusing (CIEF) has also been employed to analyze soil humic acids. This was first carried out in 1997, where three distinctive humic acid fractions were obtained [18]. To improve the resolution, Kovács and Posta modified the traditional CIEF approach by applying methyl cellulose (MeC) in the additives for electrophoresis, as well as adding polyvinyl alcohol (PVA) to protect the capillary coating [19]. The modified approach resulted in the separation of humic acids into 30–50 fractions [19].

2.2.3 Micellar electrokinetic chromatography for the separation of ionic and neutral compounds

Micellar electrokinetic chromatography (MEKC) is a modified form of capillary electrophoresis (CE) that offers several advantages over traditional CE techniques. MEKC employs micelles as the pseudo-stationary phase, instead of silica as in traditional CE. This is achieved by adding surfactants to the buffer solution at concentrations higher than the critical micellar concentration, which leads to the formation of micelles [20]. As a result, MEKC is capable of separating both ionic and neutral compounds [20], making it a versatile tool for the analysis of a wide range of analytes. MEKC was used in conjunction with preconcentration techniques to detect trace amounts of sulfonylurea herbicides in soil [21]. However, MEKC is not capable of separating larger molecules with molecular weights greater than 5 kDa [22], such as proteins and oligo-saccharides [23].

2.2.4 Microchip capillary electrophoresis for soil nutrient analysis

In studying soil composition, soil nutrient analysis is of particular importance, as it affects soil fertility, which in turn affects crop yield in agriculture. Changes in weather, water, and nutrient contents can have significant impacts on soil productivity [24]. By analyzing the nutrient content of soil, changes can be detected and addressed before they have a significant negative impact on the agricultural output [24]. CE can be used to study the inorganic nutrient contents of soil by separating ions with different sizes and charges, and the respective concentrations of these ions in the sample can then be determined by measuring their conductivity [25].

In one study, a mobile sensor was used to measure ion concentrations in water-extracted soil samples using microchip capillary electrophoresis (MCE) [25]. The ion concentrations of four different soil types, including Cambisol, Luvisol, Phaeozem, and Anthrosol, were successfully measured in the study. However, it should be noted that other ions, such as NH₄⁺, K⁺, and PO₄³⁻, may require further testing with a wider range of concentrations in the sample.

2.2.5 The advantages and limitations of CE in soil research

One of the major advantages of MCE is its high sensitivity in measuring ions, allowing for the accurate and precise quantification of nutrient concentrations in soil samples [25]. Additionally, MCE requires only a simple aqueous extraction of the sample before analysis, reducing the need for costly and time-consuming sample preparation steps. Specifically, the sample only requires shaking with deionized water and filtering through a 0.22 μm syringe filter. This simple extraction method lowers the cost and complexity of sample preparation, making CE an attractive option for routine soil nutrient analysis, especially in the field. However, MCE has limitations regarding the separation of ions under high concentrations, so further validation of its functionality in measuring a wider range of soil nutrient concentrations is needed.

3. The application of differential gradient gel electrophoresis (DGGE) and two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) in soil microbe studies

The soil microbial community consists mainly of millions of species of bacteria and fungi that perform diverse functions [26]. The microbes interact with one another as well as with plants and animals. For example, Rhizobium spp. interact with legume plants to form nitrogen-fixing nodules [27]. The diversity of soil microbes could also reflect invertebrate activities in the soil [28]. In addition, soil microbes decompose dead organisms in the soil to recycle the nutrients [29]. Studies on soil microbial communities will facilitate the understanding of the interactions among soil microbes, plants, and animals in the soil to promote ecological sustainability.

3.1 General principle of DGGE

DGGE was first utilized to test the EcoRI fragment of λ or E. coli DNA [30]. The technique has been widely employed to study microbial communities in various fields, such as medical science, marine science, and soil science [31, 32, 33]. DGGE is usually coupled with polymerase chain reaction (PCR), which amplifies marker genes such as 16S rDNA [34]. DGGE then generates a fingerprint of the amplicons for a specific microbial community.

Traditional electrophoresis separates analytes based on size differences but DGGE accomplishes this based on differences in nucleotide compositions [35]. Separation is achieved using a denaturing gel matrix for electrophoresis. Examples of denaturants include urea and formamide. By varying the concentrations of these denaturants along a gradient in the gel, different degrees of ease of strand separation can be established [33]. Compared to those with lower GC contents, DNA fragments with higher GC contents require more denaturant to achieve strand separation due to the stronger binding between G-C base pairs than A-T base pairs [36]. Since the 16S rDNA sequence varies in different microbes, the number and intensity of DNA bands observed on the gel can indicate the diversity and relative abundances, respectively, of a particular group of microbes in a soil sample [33].

3.1.1 The application of DGGE in studying soil microbial diversity

DGGE is widely used for microbial community analyses. It was first applied in ribosomal sequence analyses in a microbial community study [37]. The 16S rRNA gene encodes the small subunit ribosomal RNA of prokaryotic ribosomes, which is involved in the translation of messenger RNAs (mRNAs) into proteins to perform various biological functions. 16S rRNA is frequently analyzed in phylogenetic studies due to its conserved features and its utility as a “molecular clock” for deducing the evolutionary divergence between two living entities [38]. For example, a study analyzed soil microbial communities from different regions subjected to various agricultural management practices, such as crop rotation, and also from contaminated soil [39]. In this study, PCR amplification was performed on specific sequences in the V3 and V6/V9 regions of 16S rRNA gene in bacteria and the V3 region of 16S rRNA gene in archaea prior to DGGE analyses. The V3 region was chosen due to sequence variations that could result in distinct banding patterns in DGGE [37]. The analyses of the resulting banding patterns revealed that polyaromatic hydrocarbon (PAH)-contaminated soils had fewer bands compared to uncontaminated soils, indicating a decrease in bacterial diversity [39].

In addition to analyzing treatment-induced changes in the soil community structure, DGGE is also valuable for studying the compositions of microbes in various habitats, such as forests, and oil-contaminated paddy soil [33]. This enables the comparison of community structures between different areas. Moreover, this technique can be employed in conjunction with samples from plant tissues. For instance, DGGE was used to investigate the diversities of actinomycetes in the soil and roots of various rice cultivars [34].

Besides its applications in studying changes in community structures across different habitats and plant tissues, DGGE also allows researchers to focus on specific microbes by selecting primers to amplify targeted genes. For instance, the nifH genes, which encode nitrogen fixation–related proteins, can be analyzed by DGGE to reveal the diversity in nitrogen-fixing bacteria in rhizopheric soil [34]. By excising and sequencing the DNA bands, researchers were able to identify a Rhizobium strain among the nitrogen-fixing bacteria in the soil sample. This technique has also been employed to analyze bacteria with ammonia monooxygenase (AMO) activity and to study the diversity of fungi using 18S rDNA and internal transcribed spacer (ITS) region sequences [40, 41, 42]. The coupling with different gene amplicons allows DGGE to be a versatile tool.

3.1.2 The advantages and limitations of DGGE in soil research

One of the key advantages of DGGE is the capacity to analyze complex microbial communities in soil, allowing for the rapid investigation and comparison of community structures across ecosystems [43]. Unlike culture-based methods, DGGE enables the direct analysis of microbial communities without the need for bacterial culturing to provide a more comprehensive understanding of the community. This is especially significant as traditional culture-based methods capture only a fraction of the community, typically around 1% [44]. In addition to the microbial diversity revealed by the DNA banding patterns, DGGE also allows the excision and purification of the DNA bands for sequencing to reveal the microbial identities [45].

However, one significant limitation is the challenge in differentiating complex communities. DGGE detection is limited to targets with genome numbers larger than 10⁶ g⁻¹ dry soil [46]. It is also difficult to distinguish between soil microbial communities where different species may be present in relatively similar proportions [39]. The low DNA-focusing capability of DGGE usually results in diffused bands, which weakens its capacity to differentiate between microbial communities [33, 39]. Moreover, the heterogeneity of the genes amplified for DGGE can result in multiple bands even with a pure microbial culture [39]. The multiple bands may then lead to the misinterpretation of results.

3.2 General principle of two-dimensional polyacrylamide gel electrophoresis (2D-PAGE)

The principle behind 2D-PAGE is to separate analytes in two dimensions based on two parameters. Such a two-dimensional separation allows for a higher resolution in the separation compared to using only one parameter, that is, a one-dimensional separation. 2D-PAGE was first presented in the 1970s [47, 48] to separate proteins according to their isoelectric points (first dimension) and molecular weights (second dimension). A similar concept was adopted to separate DNA fragments based on different lengths and GC contents [42]. The separated proteins or DNA fragments can then be subjected to further analyses, such as mass spectrometry for protein identification [49] or DNA sequencing [42].

3.2.1 The applications of 2D-PAGE in analyzing soil microbial diversity

3.2.1.1 The separation of soil proteins using 2D-PAGE

2D-PAGE is a widely used technique for separating proteins within a mixture, facilitating the investigation of protein expressions in specific cell types [49] and even metaproteomics [50]. In the study by Paul and Nair, 2D-PAGE was used in conjunction with MALDI-TOF/MS (matrix-assisted laser desorption/ionization coupled with time-of-flight mass spectrometry) to study the differential protein expression patterns of a plant growth–promoting rhizobacterium (PGPR), Pseudomonas fluorescens MSP-393, under salt shock [49]. The resulting peptide mass fingerprint revealed 22 differentially regulated proteins. 2D-PAGE also enables the large-scale identification of proteins from soil. For example, more than 800 protein spots could be detected from different types of soil in a metaproteomic analysis [51]. Using 2D-PAGE coupled with MALDI-TOF/MS, and by comparing the protein profiles among untreated, mineral fertilizer–treated, and organic manure–treated paddy soils, it was found that the long-term application of pig manure promoted the functional and structural diversity of soil microbes [52].

Other than soil microbial diversity, 2D-PAGE has also been used to investigate the functional relationships between soil and plant. In a study on the rhizospheric soil of a flowering broomrape, Rehmannia glutinosa, using 2D-PAGE, 103 protein spots were successfully separated from the rhizospheric soil samples [53]. By comparing the protein expression patterns across the 2-year timeframe of R. glutinosa monoculture, differentially expressed proteins associated with the monoculture were identified. Together with the results from root exudate analyses, it was suggested that the root exudates accumulated during monoculture changed the soil microbial ecology. This study demonstrated the usefulness of 2D-PAGE coupled with MALDI-TOF/MS in the study of microbe-plant interactions.

3.2.1.2 The separation of soil DNA using 2D-PAGE

In addition to facilitating the study of protein profiles of soil microbes, 2D-PAGE can also be used to determine the soil microbial community structure through DNA separation and identification. For instance, genetic markers such as the internal transcribed spacers (ITSs) of soil microbes were amplified by PCR and separated by 2D-PAGE to reveal the operational taxonomic units (OTUs) [42]. In the study, the first dimension of separation was based on fragment lengths using a non-denaturing polyacrylamide gel while the second dimension was based on the nucleotide composition through DGGE [42]. The separation of DNA fragments based on their lengths prior to DGGE enhanced the resolution of the separation, thus enabling the detection of bacterial communities with high degrees of structural similarity [42].

3.2.2 The advantages and limitations of 2D-PAGE in soil microbe studies

After 2D-PAGE, the analyzed proteins remain intact. Such a feature allows their subsequent identification through the use of mass spectrometry. Compared to using DGGE alone, the coupling of DGGE with a non-denaturing separation of DNA samples can improve the resolution. In a study analyzing the same soil sample using DGGE alone versus 2D-PAGE coupled with DGGE, ten times more operational taxonomic units could be identified with the addition of 2D-PAGE [42].

Despite these advantages, 2D-PAGE has several limitations, such as the difficulty in reproducing results due to inconsistencies in the pH gradient with ampholytes, as well as low extraction rates of proteins with transmembrane domains and difficulties in visualizing proteins with low abundances [54]. Additionally, as protein sizes increase, such as those larger than 200–500 kDa, the efficiency of separating them on the polyacrylamide gel decreases, due to the shrinking differences in size on a logarithmic scale [30, 55].

4. The application of agarose gel electrophoresis, DGGE, and CE for soil environmental DNA analyses

Environmental DNA (eDNA) is a pool of total DNA extracted from environmental samples [56], and bulk samples containing mainly the organisms of a target taxon such as insects collected from traps [57]. eDNA is composed of mostly intracellular microbial DNA and extracellular DNA left by organisms through processes such as shedding and defecation [58]. In addition to the existence of microbes, eDNA also contains those of plants and animals that either live in or pass through that area where the sample is collected. It has been demonstrated that soil eDNA can provide rich information about the diverse community living in the terrestrial environment including microbes, plants, invertebrates, and mammals [41, 59, 60, 61].

As early as the 1980s, the presence and recovery of eDNA in different habitats, such as soil, sediment, and aquatic environment, was reported [62, 63, 64]. Distinct from traditional DNA sampling methods, eDNA is extracted directly from environmental samples instead of isolated target organisms, so it is a versatile, easy-to-perform, and non-invasive approach to collecting large-scale DNA samples of the organisms living in the habitat of interest [65].

4.1 The application of agarose gel electrophoresis in eDNA preparation and analysis

While many types of electrophoresis have been introduced in the previous sections, conventional agarose gel electrophoresis, which is a simple and effective way to separate DNA fragments, is particularly suited to soil eDNA preparation.

4.1.1 The general principle of agarose gel electrophoresis

Agarose gel electrophoresis separates DNA samples based on their lengths. Upon gelation, agarose polymers will non-covalently link with one another to form a porous network that allows DNA molecules to pass through during electrophoresis [66]. When DNA is loaded onto the agarose gel and an electric current is applied, negatively charged DNA will migrate towards the positive pole [66]. Since DNA has a relatively uniform mass-to-charge ratio, the rate of migration is mainly determined by the size of DNA [67]. Thus, DNA fragments of different molecular weights can be separated by different migration rates and distances traveled. The sizes of sample DNA can also be estimated by simultaneously running a DNA marker ladder with fragments of known sizes.

4.1.2 The applications of agarose gel electrophoresis in eDNA preparation

In addition to analyzing DNA samples based on the molecular weight, agarose gel electrophoresis is also used for soil eDNA preparation. Humic acids are often co-extracted during soil eDNA extraction. These polyphenolic compounds can interfere with enzymatic reactions including PCR and restriction digestion, reducing the utility of the extracted DNA, so it is essential to include a purification step after extraction [68]. A method known as in-gel patch electrophoresis was developed to remove humic acids by packing a chromatography patch inside an agarose gel [69]. To remove humic acids having higher molecular weights than the DNA, the sample is first applied onto the chromatography patch for electrophoresis. After some time, the electric current is reversed. At this stage, the humic acids are retained in the chromatography patch while the DNA migrates back into the agarose gel [69]. To remove humic substances with lower molecular weights than the DNA, the samples are subjected to electrophoresis until the humic acids are separated from the DNA and have migrated out of the agarose gel [69]. Compared to other techniques, including cell lysis followed by ion-exchange chromatography or conventional gel electrophoresis together with gel extraction, in-gel patch electrophoresis yields DNA with a higher purity as indicated by higher A_260/280 and A_260/230 ratios and more complete digestion by restriction enzymes [69, 70]. Agarose gel electrophoresis is also used to purify high molecular weight (HMW) DNA from crude extracts. The crude extract, loaded onto a 1% agarose gel, is subjected to electrophoresis at 20 V for 16 hours. After that, the separated HMW DNA can be purified from the gel and ligated into desired vectors for eDNA library construction [71]. This method has been used to construct soil eDNA libraries in studies investigating the access to natural product gene clusters and identifying bacterial tryptophan dimer gene clusters [72, 73].

4.1.3 The applications of agarose gel electrophoresis in eDNA quality assessment

eDNA extraction usually involves multiple steps such as grinding, homogenization, sonication, and cell lysis with sodium dodecyl sulfate (SDS). These steps may result in unintended DNA fragmentation. This shows up as smears in agarose gel electrophoresis [74]. Various eDNA extraction methods have thus been developed to avoid such mishaps. For example, the slow-stirring method yields high-purity eDNA with minimal DNA fragmentation, as visualized by agarose gel electrophoresis as a quality control step [75].

Agarose gel electrophoresis can be coupled with restriction digestion to assess the purity of the extracted eDNA. Many of the impurities in eDNA co-extracted from soil inhibit enzymatic activities. To enhance the eDNA purity, the floatation method is used to separate the soil matrix into layers to remove enzyme-inhibiting substances prior to extraction steps [76]. The extracted DNA is then subjected to restriction digestion followed by agarose gel electrophoresis to visualize the digestion products. A complete DNA digestion would mean the successful removal of enzyme inhibitors [76]. Agarose gel electrophoresis is a simple quality control step in the eDNA purification process. However, it has to be coupled with other procedures such as restriction digestion to be effective.

4.2 The applications of DGGE and CE in eDNA analyses

Soil eDNA contains a variety of DNA from different organisms, including bacteria, fungi, plants, invertebrates, and mammals [77]. Purified eDNA can be used for PCR amplification of organism-specific marker genes to selectively amplify the DNA of the organisms of interest from the soil eDNA for downstream analyses. Besides the analyses of microbial communities in soil as mentioned in the previous sections, DGGE and CE can also be applied to investigate the ecological footprints of plants and animals in the soil eDNA. For example, PCR-DGGE has been used to analyze nematode communities in soil [41]. In the study, soil eDNA extracted from various agricultural fields was used for PCR amplification of the nematode 18S rDNA using nematode-specific primer sets followed by DGGE, to analyze the nematode community in these fields by examining the different DNA banding patterns [41].

After the amplification of the target DNA, CE can be applied to titrate the PCR products, check fragment lengths, and monitor primer dimers, before high-throughput sequencing. These workflows have been employed to study various communities in the soil, including plants, insects, earthworms, and other arthropods [60, 61, 78, 79, 80]. The identification of various soil communities forms an integral part of the ecological research in different habitats.

5. Conclusion

Electrophoresis separates molecules in soil, including minerals, organic compounds, proteins, and DNA, based on their sizes and charges. The composition of soil minerals and organic compounds is an important indicator of soil quality while the protein and DNA profiles generated by electrophoresis reveal the structural biodiversity of the soil communities. Since soil is rich in microbes, electrophoretic techniques are commonly applied to study the structural diversity in soil microbial communities. Coupling them with other platforms such as mass spectrometry and DNA sequencing to identify the individual protein or DNA fragments further enhances the utility of electrophoresis in soil research. Soil protein electrophoresis has been coupled with mass spectrometry to reveal the relationships between root exudates and soil microbial community structure. Such an association enables the study of microbe-plant interactions in soil. In addition, the analyses of soil eDNA using electrophoresis can reveal the different soil communities including insects, earthworms, and other arthropods, thus promoting our understanding of different soil ecosystems. This knowledge can then be applied in ecosystem preservation and the search for beneficial interactions between different organisms in soil. The applications, advantages, and limitations of different electrophoretic techniques in soil research are summarized in Table 1.

Electrophoretic techniques	Examples of application	Advantages	Limitations	References
Denaturing gradient gel electrophoresis (DGGE)	Soil microbial community analysis	Direct and comprehensive analysis of microbial communities without the need for culturing	Blurred band patterns in analyzing complex communities	[33, 43]
Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE)	Separation of proteins in mixtures, soil community analysis	Higher resolution than one-dimensional gel electrophoresis	Difficulties in reproducing results, difficult to separate large proteins well	[51, 54]
Capillary zone electrophoresis	Humic substance and amino acid analysis	Faster analysis, simpler preparation, and no need for desalting with CE-LIF, higher resolution than chromatographic techniques	Lower sensitivity than HPLC	[15, 17]
Isoelectric focusing	Separation of proteins in mixtures	High resolution, able to concentrate target protein	Long processing time	[81]
Micellar electrokinetic chromatography	Environmental pollutant analysis	Capable of separating both ionic and neutral compounds	Difficulties in separating large molecules, such as proteins and oligo-saccharides	[20, 22]
Microchip capillary electrophoresis	On-site soil inorganic nutrient content analysis	High sensitivity, simple extraction method	Limitations on the separation of ions at high concentrations	[25]
Agarose gel electrophoresis	Soil eDNA extraction	Easy isolation of separated DNA fragments from the gel, simple and rapid way to estimate quality, size, and length of separated DNA	More precise quality check and quantification require other technologies in combination	[66]

Table 1.

Summary of the applications, advantages, and limitations of different electrophoretic techniques in soil research.

Acknowledgments

This work was supported by grants from Science, Technology and Innovation Commission of Shenzhen Municipality (Shenzhen-Hong Kong-Macau Science and Technology Program, Category C, SGDX20210823103535007) and the Hong Kong Research Grants Council: Area of Excellence Scheme (AoE/M-403/16). J.Y. Chu copy-edited this manuscript. Any opinions, findings, conclusions, or recommendations expressed in this publication do not reflect the views of the Government of the Hong Kong Special Administrative Region or the Innovation and Technology Commission.