Electrophoresis Conditions Research Articles

Single-tube Ptprc SNP genotyping of JAXBoy (CD45.1) and C57BL/6J (CD45.2) mice by endpoint PCR and gel electrophoresis

Allelic variation at the Ptprc gene, which encodes the pan-leukocyte marker CD45/Ly5, is commonly exploited to track hematopoietic reconstitution by flow cytometry in mixed bone marrow chimera transplant experiments. Historically, this was accomplished using bone marrow from C57BL/6 (Ptprcb/CD45.2/Ly5.2) and congenic B6.SJL-PtprcaPepcb/Boy (Ptprca/CD45.1/Ly5.1) mice. Recently, the Jackson Laboratory directly CRISPR-engineered the Ptprca allele in C57BL/6J mice. This new isogenic strain, termed JAXBoy, differs from wild-type C57BL/6J mice by two nucleotides, compared to the biologically significant 37 megabase (Mb) SJL interval retained in B6.SJL-PtprcaPepcb/Boy/J mice. Currently, Ptprc/CD45 variants are identified by flow cytometry or allele-specific real-time PCR, both of which require specialized workflows and equipment compared to standard genotyping of endpoint PCR products by gel electrophoresis. Here, we employed allele-specific oligonucleotides in conjunction with differential incorporation of a long non-specific oligo 5′-tail to allow for simultaneous identification of the Ptprca and Ptprcb alleles using endpoint PCR and gel electrophoresis. This method allows for integration of Ptprc genotyping into standard genotyping workflows, which use a single set of thermocycling and gel electrophoresis conditions. Importantly, the strategy of primer placement and tail addition described here can be adapted to discriminate similar single- or multi-nucleotide polymorphisms at other genomic loci.

Different behavior of Ferguson plot between agarose and polyacrylamide gels

In this study, we conducted Ferguson plot analyses using both agarose and polyacrylamide gels in native electrophoresis and SDS-PAGE. The results revealed intriguing differences in the behavior of bovine serum albumin (BSA) and other model proteins. Specifically, BSA exhibited Ferguson plot slopes that were dependent on the oligomer size in agarose native gel electrophoresis, while such size-dependent behavior was not observed in native-PAGE or SDS-PAGE. These findings suggest that Ferguson plot analysis is a suitable approach when using agarose gel under the electrophoretic conditions employed in this study. Furthermore, our investigation extended to model proteins with acidic isoelectric points and larger molecular weights, namely Ferritin and caseinolytic peptidase B (ClpB). Notably, these proteins displayed distinct Ferguson plot slopes when subjected to agarose gel electrophoresis. Intriguingly, when polyacrylamide gel was employed, ClpB exhibited multiple bands, each with its unique Ferguson plot slope, deviating from the expected behavior based on molecular size. This divergence in Ferguson plot characteristics between agarose and polyacrylamide gels points to an interesting and complex interplay between protein properties and gel electrophoresis conditions.

Affinity Capillary Electrophoresis as a Tool To Characterize Molecularly Imprinted Nanogels in Solution.

In this work, an innovative and accurate affinity capillary electrophoresis (ACE) method was set up to monitor the complexation of aqueous MIP nanogels (NGs) with model cancer-related antigens. Using α2,6'- and α2,3'-sialyllactose as oversimplified cancer biomarker-mimicking templates, NGs were synthesized and characterized in terms of size, polydispersity, and overall charge. A stability study was also carried out in order to select the best storage conditions and to ensure product quality. After optimization of capillary electrophoresis conditions, injection of MIP NGs resulted in a single, sharp, and efficient peak. The mobility shift approach was applied to quantitatively estimate binding affinity, in this case resulting in an association constant of K ≈ 106 M-1. The optimized polymers further displayed a pronounced discrimination between the two sialylated sugars. The newly developed ACE protocol has the potential to become a very effective method for nonconstrained affinity screening of NG in solution, especially during the NG development phase and/or for a final accurate quantitation of the observed binding.

Misfolding of fukutin-related protein (FKRP) variants in congenital and limb girdle muscular dystrophies.

Fukutin-related protein (FKRP, MIM ID 606596) variants cause a range of muscular dystrophies associated with hypo-glycosylation of the matrix receptor, α-dystroglycan. These disorders are almost exclusively caused by homozygous or compound heterozygous missense variants in the FKRP gene that encodes a ribitol phosphotransferase. To understand how seemingly diverse FKRP missense mutations may contribute to disease, we examined the synthesis, intracellular dynamics, and structural consequences of a panel of missense mutations that encompass the disease spectrum. Under non-reducing electrophoresis conditions, wild type FKRP appears to be monomeric whereas disease-causing FKRP mutants migrate as high molecular weight, disulfide-bonded aggregates. These results were recapitulated using cysteine-scanning mutagenesis suggesting that abnormal disulfide bonding may perturb FKRP folding. Using fluorescence recovery after photobleaching, we found that the intracellular mobility of most FKRP mutants in ATP-depleted cells is dramatically reduced but can, in most cases, be rescued with reducing agents. Mass spectrometry showed that wild type and mutant FKRP differentially associate with several endoplasmic reticulum (ER)-resident chaperones. Finally, structural modelling revealed that disease-associated FKRP missense variants affected the local environment of the protein in small but significant ways. These data demonstrate that protein misfolding contributes to the molecular pathophysiology of FKRP-deficient muscular dystrophies and suggest that molecules that rescue this folding defect could be used to treat these disorders.

Recovery of calcium and sulphur from phosphogypsum with zero by-product generation using a preparative vertical-flow electrophoresis system

Phosphogypsum has been a longstanding issue that poses environmental risks and economic pressure on society. Realizing the recycling of this waste is essential for achieving sustainable phosphorus chemical industry. In order to tackle this problem, this study examined the potential of using a preparative vertical-flow electrophoresis (PVFE) system with supporting media to recover calcium and sulphur from phosphogypsum without producing any by-products. NaOH was used to convert phosphogypsum into slaked lime and Na2SO4, with a maximum leaching efficiency of 98.6 % achieved at a NaOH concentration of 2 mol/L and a molar ratio of sodium to sulfur of 2.1. The slaked lime obtained met the quality standards outlined in the Chinese national standard (HG/T 4120–2009). Electrophoresis separation of the resulting Na2SO4 demonstrated that the NaOH effluent output could reach the desired concentration for reusing in the treatment of phosphogypsum. The addition of a solid supporting media (quartz sands) is the key to achieving the separation of Na2SO4. After considering the economic benefits, the optimal electrophoresis conditions for separating Na2SO4 were determined to be a supply voltage of 10 V and HRT of 240 min. These findings offer a directed and practical solution for the economical and effective recycling of phosphogypsum in phosphorus chemical industry.

Electrophoretic determination of chitin in insects

An electrophoretic method (on-line coupled capillary isotachophoresis with capillary zone electrophoresis with conductometric detection (cITP-CZE-COND)) for the determination of chitin in insects based on the analysis of glucosamine after acidic hydrolysis of the sample is described. Chitin is deacetylated and hydrolyzed to glucosamine by acidic hydrolysis (6 M sulfuric acid, 110 °C, 6 h). Under optimized electrophoresis conditions, glucosamine (GlcN) is separated from other sample components in cationic mode and detected with a conductometer within 15 min. The performance method characteristics of the GlcN assay, i.e., linearity (0.2–20 μmol), accuracy (103 ± 5%), repeatability (1.9%), reproducibility (3.4%), limits of detection (0.06 μmol/L) and quantification (0.2 μmol/L), were evaluated. On a series of 28 insect samples, it was proven that cITP-CZE-COND provides results of chitin content in insects comparable to the literature data. The important features of the developed cITP-CZE-COND method are easy sample treatment, high sensitivity and selectivity, and low running costs. It is clear from the above that the cITP-CZE-COND method is suitable for analysis of insect samples for chitin content.

Agarose Gel Electrophoresis-Based RAPD-PCR-An Optimization of the Conditions to Rapidly Detect Similarity of the Alert Pathogens for the Purpose of Epidemiological Studies.

Agarose gel electrophoresis is a well-known tool to detect DNA fragments amplified in polymerase chain reaction (PCR). Its usefulness has also been confirmed for epidemiological studies based on restriction fragments length polymorphism (RFLP), usually performed using pulsed-field gel electrophoresis (PFGE). Little is known on the effectiveness for alert-pathogen epidemiological studies of another less time-consuming and costly technique called randomly amplified polymorphic DNA-PCR (RAPD-PCR). Meanwhile, its usefulness is believed to be comparable to RFLP-PFGE. Therefore, the aim of the study was to establish and optimize the conditions of agarose gel electrophoresis following RAPD-PCR for 19 Enterococcus faecium strains derived from epidemic outbreaks at intensive care units. An application of different PCR primers, primer combinations, and, in particular, agarose gel concentrations and electrophoresis conditions revealed the usefulness of this relatively fast and inexpensive method based on RAPD-PCR for epidemiological studies without a compulsion to use the specialized equipment necessary for RFLP-PFGE.

Calibration of the comet assay using ionising radiation

Several trials have attempted to identify sources of inter-laboratory variability in comet assay results, aiming at achieving more equal responses. Ionising radiation induces a defined level of DNA single-strand breaks (per dose/base pairs) and is used as a reference when comparing comet results but relies on accurately determined radiation doses. In this ring test we studied the significance of dose calibrations and comet assay protocol differences, with the object of identifying causes of variability and how to deal with them. Eight participating laboratories, using either x-ray or gamma radiation units, measured dose rates using alanine pellet dosimeters that were subsequently sent to a specialised laboratory for analysis. We found substantial deviations between calibrated and nominal (uncalibrated) dose rates, with up to 46% difference comparing highest and lowest values. Three additional dosimetry systems were employed in some laboratories: thermoluminescence detectors and two aqueous chemical dosimeters. Fricke’s and Benzoic Acid dosimetry solutions gave reliable quantitative dose estimations using local equipment. Mononuclear cells from fresh human blood or mammalian cell lines were irradiated locally with calibrated (alanine) radiation doses and analysed for DNA damage using a standardised comet assay protocol and a lab-specific protocol. The dose response of eight laboratories, calculated against calibrated radiation doses, was linear with slope variance CV= 29% with the lab-specific protocol, reduced to CV= 16% with the standard protocol. Variation between laboratories indicate post-irradiation repair differences. Intra-laboratory variation was very low judging from the dose response of 8 donors (CV=4%). Electrophoresis conditions were different in the lab-specific protocols explaining some dose response variations which were reduced by systematic corrections for electrophoresis conditions. The study shows that comet assay data obtained in different laboratories can be compared quantitatively using calibrated radiation doses and that systematic corrections for electrophoresis conditions are useful.

Chromosomal polymorphism of the Ceratocystis fimbriata species complex in Brazil

Ceratocystis fimbriata is an important pathogen that causes wilt in several plant species. Despite the importance of this pathogen, knowledge about its karyotypic polymorphism and genomic architecture is limited. The main objective of this study was to investigate the karyotype of isolates of the C. fimbriata species complex from different host plants and geographical origins in Brazil. First, the identity of the isolates was confirmed conducting multilocus sequence analysis (MLSA) phylogeny using β-tubulin (TUBB), translation elongation factor 1α (TEF-1α) and mating-type (MAT1 and MAT2) gene sequences. To investigate the chromosomal polymorphism, two conditions of pulsed-field gel electrophoresis (PFGE) were used and the karyotypes of the isolates obtained. The retrotransposon-microsatellite amplified polymorphism (REMAP) molecular marker was utilized to assess the genetic variability among isolates. In the MLSA utilizing the concatenated gene sequences, Ceratocystis cacaofunesta and C. fimbriata formed separate clades, but considerable variation among C. fimbriata isolates was observed. Polymorphism in chromosome number and size was found, indicating the existence of genomic differences among isolates and occurrence of chromosomal rearrangements in the species complex. The number of chromosomes varied from seven to nine and the estimated minimum chromosome sizes were estimated to be between 2.7 and 6.0 Mbp. Small polymorphic chromosomes ranging from 1.2 to 1.8 Mbp were observed in all isolates, raising the hypothesis that they could be supernumerary chromosomes. REMAP analysis revealed a high genetic variability and that isolates from the same host tend to group together in a same cluster. Our results bring new insights into the chromosomal diversity and genome organization of the C. fimbriata complex.

Improvement of injection conditions for zone electrophoresis of proteins in capillaries coated with N, N-didodecyl-N, N-dimethylammonium bromide

Improvement of injection conditions for zone electrophoresis of proteins in capillaries coated with N, N-didodecyl-N, N-dimethylammonium bromide

Multivariate optimization of large-volume sample stacking with polarity switching by capillary electrophoresis for determination of gold nanoparticle size

Gold nanoparticles (AuNPs) are widely studied and applied due to their unique optical characteristics, such as surface plasmon resonance and extinction coefficient. In this study, large sample stacking combined with polarity switching (LVSS-PS) by capillary electrophoresis was established to determine AuNP mixtures with different particle sizes simultaneously. The multivariate experimental design containing fractional factorial design (fFD) and central composite design (CCD) was combined with LVSS-PS to estimate the optimal condition. The influence of factors on the response was evaluated by the use of the chromatography resolution statistic function to simultaneously investigate the resolution and separation under twenty-eight sets of capillary electrophoresis conditions. The results showed that the optimal condition of the separation buffer was 100 mM CAPS (pH 9.5) with 20 mM SDBS and 35 mM SDS. The separation temperature was set at 20 °C. Under this condition, four different diameters of AuNPs (5, 20, 50, and 80 nm) were successfully separated in the same run. The linear relationship between the electrophoretic mobility of AuNPs and their diameters was established, and the correlation coefficient was 0.99. Sodium citrate and gallic acid (GA) were capping agents to synthesize AuNPs. The established method was applied to determine the size of gold nanoparticles synthesized by sodium citrate and gallic acid as capping agents, respectively. The diameters of the AuNPs obtained by LVSS-PS are consistent with transmission electron microscopy.

Short- and long-term reproducibility of the COMET assay for measuring DNA damage biomarkers in frozen blood samples of the EPIC-Heidelberg cohort

The comet assay is widely used for quantification of genomic damage in humans. Peripheral blood derived mononuclear cells (PBMCs) are the most often used cell type for this purpose. Since the comet assay can be performed in an enhanced throughput format, it can be applied to large sample collections such as biobanks. The European Prospective Investigation into Cancer and Nutrition (EPIC) study is one of the largest existing prospective cohort studies, and the German Cancer Research Institute (DKFZ) in Heidelberg is a participating center with 25.000 frozen blood samples stored from around 25 years ago, enabling retrospective assessment of disease risk factors. However, experience with decades long frozen samples in the comet assay is so far missing. In Heidelberg, 800 study participants were re-invited twice between 2010 and 2012 to donate further blood samples. Here, we analyzed 299 Heidelberg-EPIC samples, compiled from frozen PBMC and buffy coat preparations selected from the different sampling time points. In addition, 47 frozen PBMC samples from morbidly obese individuals were included. For buffy coat samples, we observed a poor correlation between DNA damage in the same donors assessed at two sampling time points. Additionally, no correlation between DNA damage in buffy coat samples and PBMCs was found. For PBMCs, a good correlation was observed between samples of the same donors at the two time points. DNA damage was not affected by age and smoking status, but high BMI (>30; obesity) was associated with increased DNA damage in PBMCs. There was no indication for a threshold of a certain BMI for increased DNA damage. In conclusion, while 25 year-long stored buffy coat preparations may require adaptation of certain experimental parameters such as cell density and electrophoresis conditions, frozen PBMC biobank samples can be analyzed in the comet assay even after a decade of storage.

Optimization of electrophoresis conditions for HAp/collagen composite fiber bundles

In this study, we aimed to optimize the current conditions to maximize the tensile strength of the HAp/collagen composite fiber bundles. The HAp/collagen composite fibers prepared by the biomimetic deposition method were bundled by the electrophoresis. Their tensile strength was evaluated by the micro-tensile tests. As a result, the tensile strength of the HAp/collagen composite fiber bundles increased and then decreased with the increase of the current value. It can be considered that the higher current would accelerate both the bundling and damage of the HAp/collagen composite fibers. This tendency suggests that the optimized magnitude of the current should be determined under a trade-off relation between the bundling/orientation and the damage. Based on the results obtained in this study, it can be suggested that the optimal current value for preparing the HAp/collagen composite fiber bundles is about 4[mA].

METHOD DEVELOPMENT FOR THE DETERMINATION OF METFORMIN IN HUMAN PLASMA BY CAPILLARY ZONE ELECTROPHORESIS

Background: Metformin is an oral antidiabetic drug from the biguanide class and it is the first-line drug chosen for the treatment of type 2 diabetes. This is a drug on the list of pharmaceutical substances required to report bioequivalence study data when registering the drug. Objectives: (1) To develope a capillary zone electrophoresis method for determining metformin hydrochloride in human plasma. (2) To validate the method. Materials and methods: Human plasma, metformin hydrochloride, ranitidine hydrochloride. The method was developed and validated according to US-FDA 2018 and EMA 2011. Results: The procedure was developed by using the Agilent 7100 CE electrophoresis system with ranitidine hydrochloride as an internal standard. Sample preparation was accomplished through protein precipitation with acetonitrile. The optimal electrophoresis conditions are as follows: uncoated fused-silica capillary column of a total length of 40 cm (31.5 cm effective length, inner diameter 100 μm), phosphate buffer solution 100 mM (pH = 4), the voltage applied to both capillary ends of 15 kV, the inlet end of capillary dipped in water before sample injection, sample injection mode of 50 mBar in 7 seconds, using a PDA detector at 232 nm. The analysis method was validated according to the requirements of the US-FDA 2018 and EMA 2011 with the following criteria: system suitability with RSD < 3%; good specificity; the calibration curves were linear (r2 ≥ 0.98) in the concentration range of 0.1 – 4.0 μg/ml for metformin in human plasma; the lower limit of quantification was 0.1 μg/ml; the intra-day and inter-day accuracy were 99.41 – 105.28% and 92.47 – 106.26%, respectively; the intra-day and inter-day precision were 1.99 – 4.03% and 3.11 – 6.05%, respectively; the mean recovery of ranitidine (internal standard) was 86.2%, the mean recoveries of metformin at three levels LQC, MQC, and HQC were 72.9%, 75.9%, and 77.4%, respectively; plasma samples were stable to analysis. Conclusions: The developed method meets the requirements of US-FDA 2018 and EMA 2011 to determine metformin in plasma. Key words: Capillary zone electrophoresis, plasma, metformin

A capillary electrophoresis method for the determination of soluble monosaccharides in Ginkgo biloba leaves.

A method for the simultaneous determination of six monosaccharides by pre-column derivatization with 1-phenyl-3-methyl-5-pyrazolone and capillary electrophoresis was developed in this work. The derivatization (i.e., reaction temperature, capillary electrophoresis duration, and extraction number) and separation (i.e., pH and buffer concentration) conditions for capillary electrophoresis were optimized. Results showed that the limits of detection under optimal conditions were in the range of 0.036-0.35mg/L with a mean correlation coefficient >0.99. The recoveries were in the range of 87.3-108.49%, and the relative standard deviations of intra- and inter-day variations were in the ranges of 2.2-3.8 and 3.2-5.0%, respectively. The method was successfully applied to the analysis of six free monosaccharides in three types of Ginkgo biloba leaves.

Molecular Cloning, Expression and Macrophage Activation of an Immunoregulatory Protein from Cordyceps militaris.

Protein components of C. militaris have been reported to possess various biological activities. In our previous research, a Cordyceps militaris-derived immunoregulatory protein (CMIP) was naturally isolated and showed the activity of inhibiting the metastasis of breast cancer cells. This study aimed to obtain recombinant CMIP (rCMIP) using recombinant expression and elucidate its ability to activate macrophages. Recombinant CMIP showed one band at approximately 15 kDa or 30 kDa, or two bands at 15 kDa and 30 kDa, under different denaturation conditions of electrophoresis. The cell binding assay showed that rCMIP selectively binds to the surface of macrophages. After adhesion, it did not induce the apoptosis of RAW 264.7 cells, but promoted their proliferation. Moreover, rCMIP significantly induced the expression of M1 macrophage polarization-related molecules. The mean fluorescence intensity (MFI) of CD 86 was enhanced by 2.1-fold and 3.2-fold under 0.64 μM and 1.6 μM of rCMIP treatment, respectively. Cytokines typically expressed in M1 macrophages, such as TNF-α, iNOS, IL-6, CCL 4, CCL 5 and CXCL 10, were also considerably induced by rCMIP, while the expression of cytokines in typical M2 macrophages, like Arg-1, CCL17 and CCL22, were not changed or slightly decreased. Under rCMIP treatment, the release of NO was also appreciably induced. In the present study, we reported cloning, expression and functional characterization of rCMIP, which was naturally isolated from the fruiting body of C. militaris in our previous study. The data imply that rCMIP possesses immunomodulatory activity in macrophages.

Efficient screening for 8-oxoguanine DNA glycosylase binding aptamers via capillary electrophoresis

8-氧代鸟嘌呤DNA糖基化酶(OGG1)是人体中重要的功能蛋白,在修复DNA氧化性损伤过程中起关键作用。氧化应激等引起的氧化损伤易导致炎症反应的发生,对OGG1的抑制可以一定程度上起到缓解作用;对癌细胞OGG1的抑制有望作为癌症治疗的新方法。目前的研究多集中于小分子对OGG1功能的影响和调控,而OGG1的适配体筛选尚未见报道。作为功能配体,适配体具有合成简单、高亲和力及高特异性等优点。该文筛选了OGG1的核酸适配体,结合毛细管电泳高效快速的优点建立了两种基于毛细管电泳-指数富集进化(CE-SELEX)技术的筛选方法:同步竞争法和多轮筛选法。同步竞争法利用单链结合蛋白(SSB)与核酸库中单链核酸的强结合能力,与目标蛋白OGG1组成竞争体系,并通过增加SSB浓度来增加竞争筛选压力,以去除与OGG1弱结合的核酸序列,一步筛选即可获得与OGG1强结合的核酸序列。多轮筛选法在相同孵育条件和电泳条件下,经3轮筛选获得OGG1的核酸适配体。比较两种筛选方法的筛选结果,筛选结果中频次最高的3条候选核酸适配体序列一致,其解离常数(KD)值在1.71~2.64 μmol/L之间。分子对接分析结果表明候选适配体1(Apt 1)可能与OGG1中具有修复氧化性损伤功能的活性口袋结合。通过对两种筛选方法的对比,证明同步竞争法更加快速高效,对其他蛋白核酸适配体筛选方法的选择具有一定的指导意义。得到的适配体有望用于OGG1功能调控,以抑制其修复功能。

Rapid authentication of mono-floral honeys by capillary zone electrophoresis

Protein is the most important characteristic component of honey. The types and content of proteins in different kinds of honey are imparity, but those in the same kind of honey are very similar. Capillary zone electrophoresis (CZE) method, used to detect proteins, was developed for authenticating of four mono-floral honeys (n = 45), including medlar honey, chaste honey, jujube honey and locust honey. Electrophoresis conditions including capillary inner diameter (50–70 μm), pH (2.5–9), the concentration of anionic surfactant (0.3–0.5 mmol L−1), injection time (3–7 s) and separation voltage (3–7 KV) were optimized. The results showed that the honey sample was highly stable and the detection limit was 10%, the relative standard deviation of retention time and peak area were lower than 1.40% and 1.76%, respectively. The CZE fingerprints were established on the basis of different protein characteristics in different mono-floral honeys combined with chemometric analysis. All 45 honeys could be correctly identified according to their floral origins. This method does not need to confirm the type of protein, and has the advantages of simple and fast verification of the authenticity of honey.

Adsorption of Synthetic Dyes Under the Conditions of Capillary Zone Electrophoresis

Adsorption of Synthetic Dyes Under the Conditions of Capillary Zone Electrophoresis

Limited Proteolysis and Gel Electrophoresis in the Presence of Metal Cations: Au(III)-binding Luminescent Domain in Serum Albumins.

The purpose of the presented protocols is to determine the domain of Au(III) binding in BSA. The BSA-Au(III) compound exhibits ultraviolet (UV)-excitable red luminescence (λem = 640 nm), with unusual Stokes shifts compared to the innate UV/blue fluorescence arising from the aromatic residues. Red-luminescent complexes are formed in highly alkaline conditions above pH 10 and require a conformation change within the protein to occur. In addition, preservation of Cys-Cys disulfide bonds in BSA is necessary to obtain this red luminescence. In order to understand the mechanism of this luminescence, elucidation of the luminophore-forming Au(III) binding site is essential. A facile way to assess the luminophore-forming site would be to (1) predictably fragment the protein by enzymatic digestion, (2) react the obtained fragments with Au(III), then (3) perform gel electrophoresis to observe the well-separated fragment bands and analyze the in-gel red luminescence. However, due to the alkaline conditions and the reaction with metal cations, new limited proteolysis techniques and gel electrophoresis conditions must be applied. Particularly, the presence of metal cations in gel electrophoresis can make the band separations technically difficult. We describe this new protocol in steps to identify the red-luminophore-forming metal binding domain in BSA. This protocol can thus be applied for analyzing protein fragments that must remain in a non-denatured or a partially denatured state, in the presence of metal cations. Because the majority of proteins need metal cations to function, analyses of metal-bound proteins are often desired, which have relied on x-ray crystallography in the literature. This method, on the other hand, could be used in supplement to study the interactions of proteins with metal cations without requiring the protein crystallization and at a desired pH condition.