In Vitro Determination of Temperature-Dependent DNA Binding of the Evening Complex Using Electrophoretic Mobility Shift Assays.

Electrophoretic mobility shift assays (EMSAs) of DNA-binding proteins and labeled DNA allow the qualitative and quantitative characterization of protein-DNA complex formation using native (nondenaturing) polyacrylamide or agarose gel electrophoresis. By varying the incubation temperature of the protein-DNA binding reaction and maintaining this temperature during electrophoresis, temperature-dependent protein-DNA interactions can be investigated. Here, we provide examples of the binding of a transcriptional repressor complex called the Evening Complex, comprising the DNA-binding protein LUX ARRYTHMO (LUX), the scaffold protein EARLY FLOWERING 3 (ELF3), and the adapter protein ELF4, to its cognate DNA and demonstrate direct detection and visualization of thermoresponsive binding in vitro. As negative controls we use the LUX DNA-binding domain and LUX full length protein, which do not exhibit temperature-dependent DNA binding.

A dye elution method for the quantification of insecticidal crystal proteins from Bacillus thuringiensis and its compatibility with the presence of agro-industrial raw materials and waste products.

The insecticidal crystal proteins produced by Bacillus thuringiensis during sporulation are active ingredients against lepidopteran, dipteran, and coleopteran insects. Several methods have been reported for their quantification, such as crystal counting, ELISA, and SDS-PAGE/densitometry. One of the major tasks in industrial processes is the analysis of raw material dependency and costs. Thus, the crystal protein quantification method is expected to be compatible with the presence of complex and inexpensive culture medium components. This work presents a revalidated elution-based method for the quantification of insecticidal crystal proteins produced by the native strain B. thuringiensis RT. To quantify proteins, a calibration curve was generated by varying the amount of BSA loaded into SDS-PAGE gels. First, SDS-PAGE was performed for quality control of the bioinsecticide. Then, the stained protein band was excised from 10% polyacrylamide gel and the protein-associated dye was eluted with an alcoholic solution of SDS (3% SDS in 50% isopropanol) during 45 min at 95°C. This protocol was a sensitive procedure to quantify proteins in the range of 2.0-10.0 µg. As proof of concept, proteins of samples obtained from a complex fermented broth were separated by SDS-PAGE. Then, Cry1 and Cry2 proteins were properly quantified.

Use of PCR-DGGE-Based Molecular Methods to Analyze Nematode Community Diversity.

DGGE (denaturing gradient gel electrophoresis) is a nucleic acid separation technique applied to the evaluation of microbial biodiversity. This technique is quite rapid and cheap compared to other types of analysis. Here we describe the comparison of nematode communities inhabiting different ecosystems. After an ecologically representative sampling collection and the nematode extraction from soil, nematodes are centrifuged in Eppendorf tubes to facilitate DNA extraction. DNA from the whole community of each type of soil is extracted, amplified with primers for 18 S rDNA and used in DGGE analysis. The profiles of DGGE can be analyzed with appropriate software, and biodiversity indices can be estimated.

Supported Molecular Matrix Electrophoresis.

Distinct bands of mucins cannot be banded using a gel electrophoresis based on a molecular sieving effect due to their very large molecular weight and remarkable diversity in glycosylation. In contrast, membrane electrophoresis can separate mucins as round bands. Here, we present an analysis of mucin separation via membrane electrophoresis using a porous polyvinylidene difluoride membrane, which is highly stable against chemical modifications and various organic solvents. The separated mucins can not only be stained with dyes but also with antibodies and lectins, and glycans can be released from the excised bands and analyzed.

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.

Micropreparative Gel Electrophoresis for Purification of Nanoscale Bioconjugates.

Conventional techniques for purifying macromolecular conjugates often require complex and costly installments that are inaccessible to most laboratories. In this work, we develop a one-step micropreparative method based on a trilayered polyacrylamide gel electrophoresis (MP-PAGE) setup to purify biological samples, synthetic nanoparticles, as well as biohybrid complexes. We apply this method to recover DNA from a ladder mixture with yields of up to 90%, compared to the 58% yield obtained using the conventional crush-and-soak method. MP-PAGE was also able to isolate enhanced yellow fluorescence protein (EYFP) from crude cell extract with 90% purity, which is comparable to purities achieved through a more complex two-step purification procedure involving size exclusion and immobilized metal-ion affinity chromatography. This technique was further extended to demonstrate size-dependent separation of a commercial mixture of graphene quantum dots (GQDs) into three different fractions with distinct optical properties. Finally, MP-PAGE was used to isolate DNA-EYFP and DNA-GQD bioconjugates from their reaction mixture of DNA and EYFP and GQD precursors, samples that otherwise could not be effectively purified by conventional chromatography. MP-PAGE thus offers a rapid and versatile means of purifying biological and synthetic nanomaterials without the need for specialized equipment.

Purification and characterization of α-galactosidase isolated from Klebsiella pneumoniae in the human oral cavity.

The enzyme α-Galactosidase (α-D-galactoside galactohydrolase [EC 3.2.1.22]) is an exoglycosidase that hydrolyzes the terminal α-galactosyl moieties of glycolipids and glycoproteins. It is ubiquitous in nature and possesses extensive applications in the food, pharma, and biotechnology industries. The present study aimed to purify α-galactosidase from Klebsiella pneumoniae, a bacterium isolated from the human oral cavity. The purification steps involved ammonium sulfate precipitation (70 %), dialysis, ion exchange chromatography using a DEAE-cellulose column, and affinity monolith chromatography. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis was used to determine the molecular weight of the purified enzyme. The kinetic constants, Michaelis constant (Km) and maximal velocity (Vmax), for this enzyme were determined by using p-nitrophenyl-α-D-galactopyranoside as substrate. The results showed that the purification fold, specific activity, and yield were 126.52, 138.58 units/mg, and 21.5 %, respectively. The SDS-PAGE showed that the molecular weight of the purified enzyme was 75 kDa. The optimum pH and temperature of the purified α-galactosidase were detected at pH 6.0 and 50 °C, respectively. The kinetic constants, Michaelis constant (Km) and maximal velocity (Vmax), for this enzyme were 4.6 mM and 769.23 U/ml, respectively. α-galactosidase from Klebsiella pneumoniae was purified and characterized. (SDS-PAGE) analysis showed that the purified enzyme appeared as single band with a molecular weight of 75 kDa.

The P124A mutation of SRP14 alters its migration on SDS-PAGE without impacting its function.

SRP14 is a crucial protein subunit of the signal recognition particle (SRP), a ribonucleoprotein complex essential for co-translational translocation to the endoplasmic reticulum. During our investigation of SRP14 expression across diverse cell lines, we observe variations in its migration on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), with some cells exhibiting slower migration and others migrating faster. However, the cause of this phenomenon remains elusive. Our research rules out alternative splicing as the cause and, instead, identifies the presence of a P124A mutation in SRP14 (SRP14 P124A) among the faster-migrating variants, while the slower-migrating variants lack this mutation. Subsequent ectopic expression of wild-type SRP14 P124 or SRP14 WT and SRP14 P124A in various cell lines confirms that the P124A mutation indeed leads to faster migration of SRP14. Further mutagenesis analysis shows that the P117A and A121P mutations within the alanine-rich domain at the C-terminus of SRP14 are responsible for migration alterations on SDS-PAGE, whereas mutations outside this domain, such as P39A, Y27F, and T45A, have no such effect. Furthermore, the ectopic expression of SRP14 WT and SRP14 P124A yields similar outcomes in terms of SRP RNA stability, cell morphology, and cell growth, indicating that SRP14 P124A represents a natural variant of SRP14 and retains comparable functionality. In conclusion, the substitution of proline for alanine in the alanine-rich tail of SRP14 results in faster migration on SDS-PAGE, but has little effect on its function.

Detection and quantification of single chain rFVII impurity in final drug products by SE-UPLC and CE-SDS as an alternative to SDS-PAGE.

Recombinant factor VII, produced in recombinant BHK cell line, is secreted as a single chain zymogen form (rFVII, non-activated) in cell culture supernatant and subsequently converts to its active form during anion exchange chromatography step in the downstream purification process, with the aid of calcium ion. Single chain rFVII impurity (non-activated form) in final drug products should not exceed more than 3.0 % of total rFVIIa content. Therefore, one of the most essential quality control tests in pharmaceutical companies is to precisely quantify and report this impurity. SDS-PAGE, as a traditional method in quality control laboratories to quantify single chain rFVII, is a laborious, time-consuming, low output, and semi-quantitative method for quantification of non-activated form impurity which utilizes a densitometer to scan the gel and calculate the non-activated form band density. In this work, we developed two novel instrumental-based techniques (SE-UPLC and CE-SDS) with superior precision, accuracy, sensitivity, and efficiency that overcome SDS-PAGE shortcomings. The results of both methods were comparable to SDS-PAGE and showed an even higher correlation with expected values. Finally, we concluded that these two methods could be used as a high throughput routine method in quality control laboratories as an alternative choice to manual SDS-PAGE.

Expression and purification of epinecidin-1 variant (Ac-Var-1) by acid cleavage.

The demand for massive quantities of therapeutic active antimicrobial peptides (AMPs) is high due to their potential as alternatives to antibiotics. However, each antimicrobial peptide has unique properties, necessitating distinct synthesis and purification strategies for their large-scale production. In this study, we bio-synthesized and purified a functional enhanced variant of the AMP epinecidin-1, known as Ac-Var-1 (acid-cleavable variant-1). To generate the active peptide, we cloned the gene for Ac-Var-1 with acid-cleavable site (aspartic acid-proline) into the pET-32a expression vector, purified the fusion protein by His tag enrichment chromatography, and performed acid cleavage to release the active Ac-Var-1 peptide. After acid cleavage, the active Ac-Var-1 was purified and characterized by SDS-PAGE and mass spectrometry. The results from both techniques provided confirmation of the intactness of the purified Ac-Var-1. The Ac-Var-1 inhibited the growth of pathogenic Escherichia coli and Staphylococcus aureus. KEY POINTS : • Epinecidin-1 is a well-known antimicrobial peptide having multipotential bioactivities. • Epinecidin-1 variant is developed via the site-directed mutagenesis method to improve its structural stability and bioactivity. • AC-Var-1 development is an economical and easy method to remove peptide from tag protein.

Real-time and quantitative protein detection via polyacrylamide gel electrophoresis and online intrinsic fluorescence imaging.

The detection of intrinsic protein fluorescence is a powerful tool for studying proteins in their native state. Thanks to its label-free and stain-free feature, intrinsic fluorescence detection has been introduced to polyacrylamide gel electrophoresis (PAGE), a fundamental and ubiquitous protein analysis technique, to avoid the tedious detection process. However, the reported methods of intrinsic fluorescence detection were incompatible with online PAGE detection or standard slab gel. Here, we fulfilled online intrinsic fluorescence imaging (IFI) of the standard slab gel to develop a PAGE-IFI method for real-time and quantitative protein detection. To do so, we comprehensively investigated the arrangement of the deep-UV light source to obtain a large imaging area compatible with the standard slab gel, and then designed a semi-open gel electrophoresis apparatus (GEA) to scaffold the gel for the online UV irradiation and IFI with low background noise. Thus, we achieved real-time monitoring of the protein migration, which enabled us to determine the optimal endpoint of PAGE run to improve the sensitivity of IFI. Moreover, online IFI circumvented the broadening of protein bands to enhance the separation resolution. Because of the low background noise and the optimized endpoint, we showcased the quantitative detection of bovine serum albumin (BSA) with a limit of detection (LOD) of 20 ng. The standard slab gel provided a high sample loading volume that allowed us to attain a wide linear range of 0.03-10 µg. These results indicate that the PAGE-IFI method can be a promising alternative to conventional PAGE and can be widely used in molecular biology labs.

An easy tool to monitor the elemental steps of in vitro translation via gel electrophoresis of fluorescently labeled small peptides.

Several methods are available to visualize and assess the kinetics and efficiency of elemental steps of protein biosynthesis. However, each of these methods has its own limitations. Here, we present a novel, simple and convenient tool for monitoring stepwise in vitro translation initiated by BODIPY-Met-tRNA. Synthesis and release of very short, 1-7 amino acids, BODIPY-labeled peptides, can be monitored using urea-polyacrylamide gel electrophoresis. Very short BODIPY-labeled oligopeptides might be resolved this way, in contrast to widely used Tris-tricine gel electrophoresis, which is suitable to separate peptides larger than 1 kDa. The method described in this manuscript allows one to monitor the steps of translation initiation, peptide transfer, translocation, and termination as well as their inhibition at an unprecedented single amino acid resolution.

Extraction, Purification, and Characterization of Olive (Olea europaea L., cv. Chemlal) Polyphenol Oxidase.

Among fruits susceptible to enzymatic browning, olive polyphenol oxidase (OePPO) stood out as being unisolated from a natural source until this study, wherein we successfully purified and characterized the enzyme. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of heated and nonheated OePPO revealed distinct molecular weights of 35 and 54 kDa, respectively, indicative of its oligomeric nature comprising active and C-terminal subunits. OePPO displayed latency, fully activating with 5 mM SDS under optimal conditions of pH 7.5 and 15 °C. The enzyme demonstrated monophenolase activity and showcased the highest efficiency toward hydroxytyrosol. Despite its low optimal temperature, OePPO exhibited high thermal resistance, maintaining stability up to 90 °C. However, beyond this threshold, the oligomeric enzyme disassociated, yielding a denatured main subunit and C-terminal fragments. Six OePPO genes were found in the fruits. Tryptic digestion identified the enzyme as mature OePPO1 (INSDC OY733096), while mass spectrometry detected the active form mass alongside several C-terminal fragments, revealing potential cleavage sites (Gly407, Tyr408).

Protein profile pattern analysis: A multifarious, in vitro diagnosis technique for universal screening.

Universal health care is attracting increased attention nowadays, because of the large increase in population all over the world, and a similar increase in life expectancy, leading to an increase in the incidence of non-communicable (various cancers, coronary diseases, neurological and old-age-related diseases) and communicable diseases/pandemics like SARS-COVID 19. This has led to an immediate need for a healthcare technology that should be cost-effective and accessible to all. A technology being considered as a possible one at present is liquid biopsy, which looks for markers in readily available samples like body fluids which can be accessed non- or minimally- invasive manner. Two approaches are being tried now towards this objective. The first involves the identification of suitable, specific markers for each condition, using established methods like various Mass Spectroscopy techniques (Surface-Enhanced Laser Desorption/Ionization Mass Spectroscopy (SELDI-MS), Matrix-Assisted Laser Desorption/Ionization (MALDI-MS), etc., immunoassays (Enzyme-Linked Immunoassay (ELISA), Proximity Extension Assays, etc.) and separation methods like 2-Dimensional Polyacrylamide Gel Electrophoresis (2-D PAGE), Sodium Dodecyl-Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE), Capillary Electrophoresis (CE), etc. In the second approach, no attempt is made the identification of specific markers; rather an efficient separation method like High-Performance Liquid Chromatography/ Ultra-High-Performance Liquid Chromatography (HPLC/UPLC) is used to separate the protein markers, and a profile of the protein pattern is recorded, which is analysed by Artificial Intelligence (AI)/Machine Learning (MI) methods to derive characteristic patterns and use them for identifying the disease condition. The present report gives a summary of the current status of these two approaches and compares the two in the use of their suitability for universal healthcare.

High-resolution nucleic acid detection using online polyacrylamide gel electrophoresis platform.

Polyacrylamide gel electrophoresis (PAGE) is one of the most popular techniques for the separation and detection of nucleic acids. However, it requires a complicated detection procedure and offline detection format, which inevitably leads to band broadening and thus compromises the separation resolution. To overcome this problem, we developed an online PAGE (OPAGE) platform by integrating the gel electrophoresis apparatus with the gel imaging system, so as to obviate the need for the complicated detection procedure. Notably, OPAGE enabled the real-time monitoring of the separation process and the immediate imaging of the separation results once the electrophoresis ended. Using a series of synthetic DNAs with different lengths as samples, we demonstrated that the OPAGE platform enhanced 32-64 % of the number of theoretical plates, showed a robust dynamic range of 0.1-12.5 ng/µL, and realized a limit of detection as low as 0.08 ng/µL DNA. Based on our results, we anticipate that the OPAGE platform is a promising alternative to traditional nucleic acid gel electrophoresis for simple and high-resolution detection and quantification and nucleic acid.

SURE gel electrophoresis: A method for improved detection and purification of dilute nucleic acid samples.

Agarose gel electrophoresis is performed routinely by molecular biologists as both an analytical and a preparative method for characterization of nucleic acids. Gel analysis of highly dilute DNA solutions is challenging because of the limited sensitivity of detection available with conventional methods. In this study a new approach is described for concentrating samples directly within gels called SURE (successive reloading) electrophoresis. The approach involves loading of dilute samples multiple times into a single well, with each loading followed by a brief pulse of electrical current before the next sample is loaded. The procedure generates single bands created by molecular stacking that exhibit strongly enhanced signal intensities and minimal band broadening. Using optimized voltages and time intervals as many as 20 successive loadings could be performed and up to 800 µL could be loaded into a single well. Gel extraction and fluorescent quantitation demonstrated that approximately 97 % of the DNA from each loading was incorporated into the resultant band. Highly dilute DNA samples (<0.0007 ng per microliter) could be readily detected after six loadings. The method produced good results with either TAE or TBE as electrophoresis buffers, using loading dyes with or without SDS, and in both minigels and large gels.

Centrifugal Gel Crushing Tips for Gel-Based Proteome Analysis.

We have developed a centrifugal gel-crushing method using a pipet tip. Polyacrylamide gel slices are extruded from the narrowing cavity of a pipet tip by centrifugation in a few minutes to crush them into pieces of appropriate size. The size of the crushed gel could be controlled by several parameters, including centrifugal force and pipet tip cavity. In shotgun proteomics, gel-based LC/MS/MS, so-called GeLC/MS/MS, involves the essential but tedious processes of prefractionation by SDS-PAGE, followed by dicing the entire gel lane into several parts, fine dicing, and in-gel digestion after the diced gel is manually transferred to a microtube. In this study, we developed an alternative way to crush the prefractionated gel slice into optionally small and irregular-shaped gels by centrifugal extrusion of the sliced gel from the narrow cavity of a pipet tip. As a result, we observed improved recovery and reproducibility of digested proteins compared to the conventional method of manual dicing. We believe that this simple and rapid method of crushing polyacrylamide gels, which allows for parallel operations and automation, is useful for GeLC/MS/MS analysis and applicable to other approaches, including top-down proteomics.

Germplasm characterization and SDS-PAGE analysis of caper (Capparis spinosa L.) from different provenances.

BACKGROUND: Capparis spinosa L. is a typical desert plant that is resistant to high temperatures and drought, and at the same time is rich in medicinal and food values. The objective of this study is to explore the variations in nutrient composition, morphological characteristics, and SDS-PAGE patterns of caper seeds from different provenances, aiming to provide insights for the selection of superior seed provenances. RESULTS: In this experiment, there were significant differences in the morphological characteristics and major nutritional components of caper seeds from different provenances. Seeds from the YKL (Karayagaqi Township, Yining County) and YKG (G218, KashiTown, Yining County) regions were larger in size compared to seeds from other regions. Among the four measured nutritional components, crude fat had the highest content, especially in the YKL and YKG region. The results of correlation analysis showed that crude fat was negatively correlated with soluble sugar and soluble protein but significantly positively correlated with starch content. As longitude increased from east to west, the morphological characteristics gradually increased. Based on the principal component analysis of all the parameters of the seeds, the eight provenances could be classified into three groups. HM (Hami), TGS (S202, Gaochang District, Turpan), HYW (Wubao Town, Yizhou District, Hami), TQQ (Qiquanhu Town, Turpan), and TLF (Turpan) were a group with higher soluble protein, soluble sugar, and water content. YKL and YKG were in one group, which had larger seed grains with high crude fat and starch content. AKS (Aksu) was in a separate group. The protein fractions from seeds of eight regions were extracted using Osborne fractionation method, it was found that glutelin content was the highest, while albumin content was the lowest. After these proteins were analyzed by SDS-PAGE, the electrophoretic patterns showed that the protein molecular weights were relatively small, and there were differences in protein bands among different provenances. CONCLUSION: According to the PCA results, the eight seed provenances could be divided into three groups. There were both geographically distant ones clustered into one group, and those close to each other were also divided into one group. There were differences in seed morphology, nutrient content and SDS-PAGE profiles among the different seed sources. This difference might be caused by a combination of geographic and climatic factors. In addition, YKL and YKG were roughly selected as good seed provenances, which provided a theoretical basis for the development of C. spinosa L. germplasm resources.

Ferritin microheterogeneity, subunit composition, functional, and physiological implications.

Ferritin is a ubiquitous intracellular iron storage protein that plays a crucial role in iron homeostasis. Animal tissue ferritins consist of multiple isoforms (or isoferritins) with different proportions of H and L subunits that contribute to their structural and compositional heterogeneity, and thus physiological functions. Using size exclusion and anion exchange chromatography, capillary isoelectric focusing (cIEF), and SDS-capillary gel electrophoresis (SDS-CGE), we reveal for the first time a significant variation in ferritin subunit composition and isoelectric points, in both recombinant and native ferritins extracted from animal organs. Our results indicate that subunits composition is the main determinant of the mean pI of recombinant ferritin heteropolymers, and that ferritin microheterogeneity is a common property of both natural and recombinant proteins and appears to be an intrinsic feature of the cellular machinery during ferritin expression, regulation, post-translational modifications, and post-subunits assembly. The functional significance and physiological implications of ferritin heterogeneity in terms of iron metabolism, response to oxidative stress, tissue-specific functions, and pathological processes are discussed.

A single step and rapid protein extraction protocol developed for cell lines and tissues: Compatible for gel based and gel free proteomic approaches.

Proteins are crucial research molecules in modern biology. Almost every biological research area needs protein-based assays to answer the research questions. The study of the total protein content of a biological sample known as Proteomics, is one of the highly rated qualitative and quantitative approach to address numerous biological problems including clinical research. The key step to successfully generate high quality proteomics data is the efficient extraction of proteins from biological samples. Although different methods are in use for protein extraction from a wide variety of samples, however, because of their prolonged protocol and multiple steps involved, final protein yield is sacrificed. Here, we have shown the development of a simple single step method for extraction of proteins from mammalian cell lines as well as tissue samples in an effective and reproducible manner. This method is based on lysis of samples directly in a modified lysis buffer without CHAPS (7 M Urea, 2 M Thiourea, and 10 mM Tris-Cl; pH 8.5) that is compatible with gel based and gel free approaches. This developed protocol is reliable and should be useful for a wide range of proteomic studies involving various biological samples.