CONCLUSIONS: LAMP assay is widely used for detecting pathogens. We observed that the conventional and gradient polymerase chain reaction (PCR) could not detect the extracted Escherichia coli DNA; real-time PCR was able to detect up to a certain limit (10-8 bacterial dilution). At the same time, the LAMP assay could detect the bacteria at a much lower concentration (10-14 dilution). The results of the LAMP assay were evaluated using agarose gel electrophoresis and DNA binding dye (PicoGreen), but only gel electrophoresis gave reliable results. Therefore, we propose using electrophoresis-based amplicon detection to overcome the limitations of dye-based detection. We believe that this amplicon detection will go a long way in the screening of potable drinking water.

The white stork (Ciconia ciconia) is a ciconiiform species widely represented in zoological institutions. Plasma protein electrophoresis is widely used in avian patients for assessment of inflammatory conditions, but reference intervals for this testing modality are lacking for the white stork. The two main electrophoretic methods are agarose gel electrophoresis (AGE) and capillary zone electrophoresis (CZE). This study assessed fresh plasma samples of healthy adult white storks (n = 30). Statistical analyses were performed to evaluate agreement between AGE and CZE. Typical electrophoretic fractions were obtained from both methods (prealbumin, albumin, α1, α2, β, γ1, and γ2). The AGE and CZE methods were not equivalent for determining major electrophoretic fractions (except β-globulins) and albumin:globulin ratio on plasma samples. An additional prealbumin fraction was seen with CZE. Reference intervals were established for each method as the smallest n group was 27 individuals for a given value; most values had normal distribution, and robust or parametric methods were used on the data.

UNASSIGNED: Knowledge of reference intervals for blood analytes, including serum protein fractions, is of great importance for the identification of infectious and inflammatory diseases and is often lacking in wild animal species.
UNASSIGNED: Serum samples were obtained from European minks enrolled in the breeding program (n = 55). Agarose gel electrophoresis (AGE) and capillary zone electrophoresis (CZE) were used to separate and identify protein fractions. Albumin, α1, α2, β, and γ-globulins fractions were identified in all mink sera by both electrophoresis methods. Reference intervals (90% CI) were determined following the 2008 guidelines of the Clinical Laboratory Standard Institute. The methods were compared using Passing-Bablok regression, Bland-Altman analysis, and Lin\'s concordance correlation.
UNASSIGNED: A significant bias was found between methods for α1, α2, and γ-globulin. Lin\'s concordance correlation was considered unacceptable for α1, α2, and β-globulins. Differences for gender between methods were found for albumin and α2-globuins, which were higher for males than females. γ-globulins were higher for adults than young minks using both methods; however, α1 and α2-globulins were lower.
UNASSIGNED: Both methods are adequate for identifying serum protein disorders, but the AGE and CZE methods are not equivalent. Therefore, reference intervals for each technique are required.

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.

The present study is focused on application of a natural compound, 3, 5-dihydroxy 4\', 7-dimethoxyflavone (DHDM) from a medicinal plant Alpinia nigra for nucleic acid detection and differential cell staining. DHDM was found to interact with nucleic acid and forms complex, which was investigated for various applications. It was successfully utilized to visualize plasmid, genomic, and ds-linear DNA in agarose gel electrophoresis without affecting the DNA mobility in the gel. Fluorescence of DHDM increased several fold upon binding to dsDNA. Photostability of the compound was assessed and showed photobleaching effect that decreased gradually over time. Application of the compound was further extended to differential cell staining. When observed in fluorescence microscope, DHDM stained the dead cells and differentiated them from live cells in the case of bacterial, yeast, and mammalian cells. Higher concentration of the compound was found to be less cytotoxic to cancerous cells. Nucleic acid staining dyes like Ethidium bromide (EtBr), Propidium iodide (PI), etc. are carcinogens and environmental pollutants and therefore DHDM a natural compound, is a major benefit and thus can serve as an alternative to the current dyes.

Organoids are 3D cell cultures with microanatomies mimicking aspects of real organs, useful for e.g. animal-free studies of development, disease, and drug discovery. The cell medium of organoid models of Langerhans islets, regulating blood glucose levels by insulin secretion, can be analyzed by liquid chromatography-mass spectrometry (LC-MS). However, organoid medium complexity is a major challenge, as matrix interferences can reduce sensitivity and selectivity, even with optimized LC-MS conditions. By applying preparative agarose gel electrophoresis-electrodialysis (PGE-ED), we were able to decrease the cell medium background signal, allowing for reduced interferences affecting LC-MS analysis of human insulin.

Trivalent lanthanide ions are known for their ability to interact with calcium-binding sites in various proteins. There is a need to assess the bioavailability of lanthanides and other heavy metals introduced into the body as components of implants or as contrast agents. This study aimed to develop a method to address bioavailability and/or presence of trivalent lanthanide ions by examining electrophoretic mobility in an agarose gel of a plasmid harboring the human metallothionein-II gene (hMT-II). Mobility of the plasmid was specifically altered by a monoclonal antibody raised against the zinc-binding transcription factor that controls the activity of the hMT-II gene. This study showed that the plasmid acquired a lanthanide-specific mobility pattern that allowed the presence of lanthanide ions to be readily determined in a 0.8% agarose gel. These findings suggest that this plasmid/monoclonal antibody combination under selected conditions may be useful in industrial, environmental, and biomedical settings to identify, separate, or capture lanthanide ions in complex mixtures that contain an array of metal ions.

Long-read sequencing technologies require high-molecular-weight (HMW) DNA of sufficient purity and integrity, which can be difficult to obtain from complex biological samples. We propose a method for purifying HMW DNA that takes advantage of the fact that DNA\'s electrophoretic mobility decreases in a high-ionic-strength environment. The method begins with the separation of HMW DNA from various impurities by electrophoresis in an agarose gel-filled channel. After sufficient separation, a high-salt gel block is placed ahead of the DNA band of interest, leaving a gap between the separating gel and the high-salt gel that serves as a reservoir for sample collection. The DNA is then electroeluted from the separating gel into the reservoir, where its migration slows due to electrostatic shielding of the DNA\'s negative charge by excess counterions from the high-salt gel. As a result, the reservoir accumulates HMW DNA of high purity and integrity, which can be easily collected and used for long-read sequencing and other demanding applications without additional desalting. The method is simple and inexpensive, yields sequencing-grade HMW DNA even from difficult plant and soil samples, and has the potential for automation and scalability.

Staining and visualization of the nucleic acid bands on agarose gels using ethidium bromide (EB) has been a widely used technique in molecular biology. Although it is an efficient dye for this purpose, EB is known to be mutagenic and genotoxic in humans. This led to the emergence of various alternative dyes, which were claimed to be safer and more efficient than EB. However, these dyes portray varied sensitivity and interference with the electrophoretic mobility of nucleic acids. This work aimed at assessing ten nucleic acid-binding dyes and two prestained dyes for these properties by three staining techniques, such as precasting, preloading, and poststaining. Of these, preloading was not suitable for any of the dye while poststaining worked optimal for most of them. Precasting was suitable for only four dyes viz. DNA Stain G, SYBR™ safe, EZ-Vision® in-gel, and LabSafe™. Poststaining was, in general, a costlier method than precasting. The work gives a comprehensive understanding of the performance of nucleic acid-binding dyes for routine molecular biology experiments.

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.