Urea-polyacrylamide Gel Electrophoresis Research Articles

Melittin alcalase-hydrolysate: a novel chemically characterized multifunctional bioagent; antibacterial, anti-biofilm and anticancer.

The prevalent life-threatening microbial and cancer diseases and lack of effective pharmaceutical therapies created the need for new molecules with antimicrobial and anticancer potential. Bee venom (BV) was collected from honeybee workers, and melittin (NM) was extracted from BV and analyzed by urea-polyacrylamide gel electrophoresis (urea-PAGE). The isolated melittin was hydrolyzed with alcalase into new bioactive peptides and evaluated for their antimicrobial and anticancer activity. Gel filtration chromatography fractionated melittin hydrolysate (HM) into three significant fractions (F1, F2, and F3), that were characterized by electrospray ionization mass spectrometry (ESI-MS) and evaluated for their antimicrobial, anti-biofilm, antitumor, and anti-migration activities. All the tested peptides showed antimicrobial and anti-biofilm activities against Gram-positive and Gram-negative bacteria. Melittin and its fractions significantly inhibited the proliferation of two types of cancer cells (Huh-7 and HCT 116). Yet, melittin and its fractions did not affect the viability of normal human lung Wi-38 cells. The IC50 and selectivity index data evidenced the superiority of melittin peptide fractions over intact melittin. Melittin enzymatic hydrolysate is a promising novel product with high potential as an antibacterial and anticancer agent.

An open-source, 3D printed inkjet DNA synthesizer

Synthetic oligonucleotides have become a fundamental tool in a wide range of biological fields, including synthetic biology, biosensing, and DNA storage. Reliable access to equipment for synthesizing high-density oligonucleotides in the laboratory ensures research security and the freedom of research expansion. In this study, we introduced the Open-Source Inkjet DNA Synthesizer (OpenIDS), an open-source inkjet-based microarray synthesizer that offers ease of construction, rapid deployment, and flexible scalability. Utilizing 3D printing, Arduino, and Raspberry Pi, this newly designed synthesizer achieved robust stability with an industrial inkjet printhead. OpenIDS maintains low production costs and is therefore suitable for self-fabrication and optimization in academic laboratories. Moreover, even non-experts can create and control the synthesizer with a high degree of freedom for structural modifications. Users can easily add printheads or alter the design of the microarray substrate according to their research needs. To validate its performance, we synthesized oligonucleotides on 144 spots on a 15 × 25-mm silicon wafer filled with controlled pore glass. The synthesized oligonucleotides were analyzed using urea polyacrylamide gel electrophoresis.

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.

The modification of nisin with homocysteine thiolactone and its effect on antimicrobial activity.

The aim of the present study is to make an important contribution to the literature by focusing on the preparation of the N-homocysteine conjugate of nisin and evaluating the effect of the N-homocysteinylation reaction on its antimicriobial activity. The modification process was monitored using both acetic acid urea polyacrylamide gel electrophoresis (AAU-PAGE) and tricine sodium dodecyl sulphate polyacrylamide gel electrophoresis (tricine SDS-PAGE). The antibacterial effectiveness of modified nisin was assessed against Staphylococcus aureus ATCC 6538, Enterococcus faecium ATCC 9097, Bacillus subtilis ATCC 6633, Lactococcus lactis ssp. cremoris AÜ, Listeria monocytogenes NCTC 5348, and Escherichia coli RSKK. Optimal conditions for achieving the highest N-homocysteinylation degree (6.30%) were determined as 6 mg/mL nisin, 150 mM homocysteine thiolactone, 150 rpm shaking rate, pH of 3.0, and a reaction time of 6 h. The modified nisin obtained did not have a significant inhibitory effect on the strains tested except E. faecium. E. faecium was inhibited by the modified nisin and its antibacterial activity was determined as approximately 10% of the antibacterial activity of unmodified nisin. On the other hand, hydrolysis of nisin by trypsin and thermolysin resulted in significant specific side chain modifications induced by the homocysteine-thiolactone reaction, especially at Lys12 and Lys22. The results provide valuable insights into the potential of N-homocysteinylation to improve the antibacterial properties of nisin and also suggest that the effects of specific modifications identified during the modification process should be investigated.

Holo/apo conversion two-dimensional urea PAGE for speciation of Fe3+-bound transferrin in serum.

This paper presents holo/apo conversion two-dimensional urea polyacrylamide gel electrophoresis (HAC-2D urea PAGE) as a novel method for speciating Fe3+-bound transferrin (Tf) species in biological samples, with a combination of metal ion contaminant sweeping (MICS) technique and Fe3+ detection PAGE. In the HAC-2D urea MICS-PAGE approach, HAC was performed to dissociate all the Fe3+ ions bound to Tf from the Fe-Tf species, during a two-step urea PAGE. Using this method, Fe2-Tf, FeN-Tf, and FeC-Tf (holo-Tf, Fe3+-bound Tf attached to N-lobe, and Fe3+-bound Tf attached C-lobe, respectively) were completely isolated based on the difference in the higher-order structure of Tf, visible as horizontally aligned spots off the diagonal. The Fe3+ ions bound to Tf in each gel fraction were determined using PAGE with a fluorescent probe. Without the MICS technique, which electrophoretically removes all contaminant Fe3+ ions from the gel medium to ensure accurate determination of the Fe3+ concentration, it becomes challenging to precisely measure the distribution of metalloprotein species owing to the contaminants. Finally, the distribution of each Fe-bound Tf in a standard human serum sample was successfully determined by complete separation from large amounts of coexisting proteins, and the free Fe3+ concentration in the serum was estimated.

Milk-Clotting Properties and Primary Proteolysis of a Lyophilised Extract from Artichoke Flowers (Cynara scolymus L.).

A few studies have investigated Cynara scolymus enzymes as a substitute for calf rennet in cheese making. They used aqueous extracts prepared by maceration of plant material and stored by freezing. However, it was indicated that lyophilisation is a better alternative to preserve the coagulant properties of plant extracts over a longer period of time, as it is a more controllable, stable and hygienic alternative with a better shelf life that is easier to transport, store and standardise. We obtained a lyophilised extract of mature artichoke flowers, named CS, which was characterised for its milk-clotting properties at different pH and temperatures. In addition, the potential yield, whey composition and the primary hydrolysis profile of caseins by urea-polyacrylamide gel electrophoresis (PAGE) of mini curds prepared with different doses of coagulant were determined. The lyophilised extract was able to hydrolyse casein and showed stable proteolytic activity at pH=6.4 and 37 °C for 50 min, which decreased when the process temperature was increased to 41 and 45 °C and was lost at 70 °C. On the other hand, milk-clotting activity increased significantly (p<0.001) when the temperature increased from 37 to 45 °C and the pH of the milk decreased from 6.8 to 5.8. Potential yield ​​between 10 and 17 % was obtained for the mini curds prepared with the lyophilised artichoke extract, and the casein degradation pattern obtained by urea-PAGE was similar to that of the commercial coagulant. On a laboratory scale, our work has shown that the lyophilised artichoke extract has sufficient proteolytic and coagulant activity to be used as a milk coagulant, i.e. plant rennet, in cheese making as an alternative to animal rennet. As this extract is lyophilised, it has the advantage of being a better alternative in terms of preservation and shelf-life. It offers an innovative way to diversify cheese products and appeal to consumers with different dietary preferences and needs.

Determination of the effects of proteolysis-based changes by adjunct lactobacilli on the bioactivity (ACE-inhibitory and antioxidant activities) of cheese: a model cheese study.

Bioactive properties, proteolysis and microbiology of model cheeses with and without adjunct lactobacilli (Lactobacillus helveticus, Lactiplantibacillus plantarum, Lactobacillus bulgaricus and L. casei) were studied during 120 days of storage at 8 or 16°C. Bioactive properties were observed in peptide fractions (< 3kDa, 3-10kDa, < 10kDa) separated using ultrafiltration membranes. Antioxidant activity of these fractions was determined by radical scavenging assays as ABTS [2, 2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid)]. Angiotensin-converting enzyme-inhibitory (ACE-i) activity (% and IC50) and peptide profiles of 70% ethanol-soluble and -insoluble fractions were determined by RP-HPLC. Use of lactobacilli as an adjunct culture significantly changed the RP-HPLC peptide profiles of the cheeses; however, slight changes were observed in the patterns of urea-polyacrylamide gel electrophoresis. Fractions smaller than 3kDa had higher ACE-i and antioxidant activities for all cheese samples. In conclusion, this study indicates that the addition of lactobacilli as an adjunct culture contributed to the formation of bioactive compounds in the model cheeses and also changed the proteolysis levels.

The effect of dephosphorylation on the properties of αS1-casein enriched protein

αS1-Casein was dephosphorylated using alkaline phosphatase without the interference from proteolysis. The progressive dephosphorylation was monitored by alkaline urea polyacrylamide gel electrophoresis and mass spectrometry techniques. Little change was observed in the SDS-polyacrylamide gel patterns for the native and dephosphorylated αS1-casein. Dephosphorylation modified the zeta-potential of the αS1-casein, shifting the pH versus zeta potential curves to higher pH as dephosphorylation increased. The isoelectric pH of the dephosphorylated αS1-casein increased by about 0.55 pH units when compared with the native protein. Dephosphorylation increased the surface hydrophobicity of αS1-casein and a negative linear correlation was observed between the surface hydrophobicity and the isoelectric pH of αS1-casein. Dephosphorylation did not affect the secondary structure of the αS1-casein.

Low Resource Integrated Platform for Production and Analysis of Capped mRNA.

The existing platform for large-scale mRNA production is fast, but consumable costs, process technicality, and complexity represent key bottlenecks limiting global mRNA biologics manufacturing. Another challenge is the lack of a consolidated platform for mRNA product characterization and assays that meet regulatory requirements. Bridging these innovation gaps to simplify processes and reduce cost would improve mRNA biologics manufacturability, especially in low-resource settings. This study develops a "cotranscriptional" capping strategy that utilizes T7 RNA polymerase, and the Vaccinia Capping System to synthesize and cap mRNA. We created an "integrated reaction buffer" that supports both capping enzymes for catalytic and in vitro transcription processes, enabling one-pot, two-step capped mRNA synthesis. Additionally, we report a novel, one-step analytic platform for rapid, quantitative, capped mRNA analysis. The assay involves target mRNA segment protection with cheap DNA primers and RNase digest of non-hybridized or non-target sequences before analysis by single nucleotide-resolving urea-polyacrylamide gel electrophoresis (PAGE). The integrated approach simplifies production processes and saves costs. Moreover, this assay has potential applications for mRNA analyses and post-transcriptional modification detection in biological samples. Finally, we propose a strategy that may enable unparalleled sequence coverage in RNase mass mapping by adapting the developed assay and replacing urea-PAGE with mass spectrometry.

A SYBR Gold-based Label-free in vitro Dicing Assay.

In Arabidopsis, DICER-LIKE PROTEIN 3 (DCL3) cuts the substrate pre-siRNA into a product siRNA duplex, encompassing one 23-nt strand and one 24-nt strand. To monitor the separation of the siRNA duplex with only 1-nt difference, we developed this protocol to evaluate the in vitro dicing activity of DCL3. The method can be applied for measuring the lengths of single-stranded RNA separated through denaturing urea polyacrylamide gel electrophoresis (urea PAGE), which are visualized by a label-free fluorescence SYBR Gold, and quantified in a multi-function imager. This label-free method is easy to conduct, has low cost, and lacks the hazard of the traditional radio-labeled method. This method can also be adapted to the other Dicers and small RNAs.

Stability and Activity of the 10-23 DNAzyme Under Molecular Crowding Conditions.

DNAzymes are biocatalysts that have been selected in vitro and their function inside cells (in vivo) is extremely low. Thus, almost all studies have been carried out in diluted solutions (in vitro). The cellular presence of molecules such as amino acids, polypeptides, alcohols, and sugars introduces forces that modify the kinetics and thermodynamics of DNAzyme-mediated catalysis. The crowded intracellular environment referred to as molecular crowding can be mimicked by adding high concentrations of natural or synthetic macromolecules to the reaction conditions. Here, we investigate the activity of the 10-23 DNAzyme and the stability of the DNAzyme:RNA complex under molecular crowding conditions. Therefore, we use a Förster resonance energy transfer (FRET)-based activity assay in combination with denaturing urea polyacrylamide gel electrophoresis and circular dichroism (CD) spectroscopy.

Urea Gel Electrophoresis in Studies of Conformational Changes of Transferrin on Binding and Transport of Non-Ferric Metal Ions.

Transferrin (Tf) is a crucial transporter protein for Fe(III), but its biological role in binding other metal ions and their delivery into cells remain highly controversial. The first systematic exploration of the effect of non-Fe(III) metal ion binding on Tf conformation has been performed by urea-polyacrylamide gel electrophoresis (urea-PAGE), which is commonly used for nucleic acids but rarely for proteins. Closed Tf conformation, similar to that caused by Fe(III)-Tf binding, was formed for In(III), V(III) or Cr(III) binding to Tf. In all these cases, metal distribution between Tf lobes and/or the rate of metal release under acidic conditions differed from that of Fe(III)-Tf. By contrast, Ga(III) and V(IV) did not form closed Tf conformation under urea-PAGE conditions. Apart from Fe(III), only In(III) was able to increase the proportion of closed Tf conformation in whole serum. These results suggest that Tf is unlikely to act as a natural carrier of any metal ion, except Fe(III), into cells but can reduce toxicity of exogenous metal ions by binding them in serum and preventing their entry into cells.

Predicting adulteration of grated Parmigiano Reggiano cheese with Ricotta using electrophoresis, multivariate nonlinear regression and computational intelligence methods

Urea polyacrylamide gel electrophoresis combined with statistical methods was used to detect the addition of Ricotta to grated Parmigiano Reggiano cheese. The adulterated samples were prepared by adding grated Ricotta to grated Parmigiano Reggiano cheese in six different ratios. The logistic regression and rank correlation performed worse than the naïve Bayes classifier at identifying samples of grated Parmigiano Reggiano containing more than 20% Ricotta in terms of area under the receiver operating characteristic curve and classification accuracy. This work can be considered a pilot study to investigate the potential of machine‐learning methods to predict the authenticity of Parmigiano Reggiano cheese.

Validation of a reversed‐phase high‐performance liquid chromatographic method for the quantification of primary proteolysis during cheese maturation

A new fast and reliable reversed‐phase high‐performance liquid chromatographic method is proposed for quantifying all the major casein fractions during cheese proteolysis as an alternative to more complex methodologies. Excellent separation of the caseins, para‐κ‐casein (ρ‐κ‐CN), αs1‐casein (αs1‐CN), αs1‐I‐casein (αs1‐I‐CN), αs2‐casein (αs2‐CN), and β‐casein (β‐CN) was achieved, in terms of linearity, reproducibility, repeatability, accuracy and certified standard recovery. Validation by quantification of semihard and Maasdam‐style cheeses gave comparable results for ρ‐κ‐CN, β‐CN and αs‐casein (αs‐CN) to published data for both urea–polyacrylamide gel electrophoresis and capillary electrophoresis.

Zinc fractionation in cow, goat, sheep and soybean milk samples using gel-electrophoresis and determination by electrothermal atomic absorption spectrometry (ETAAS)

A screening method for zinc levels in different milk samples (raw cow, raw sheep, UHT cow, UHT goat and soybean milk base) was performed to establish the Zn levels’ differences in protein samples. The samples were digested in a cavity microwave oven and the total Zn levels in the extracts were determined by flame absorption atomic spectrometry (FAAS). The protein separation was performed by urea polyacrylamide gel electrophoresis (UREA-PAGE). Protein bands were digested in the cavity microwave oven and Zn-protein analysis was further conducted by electrothermal atomic absorption spectrometry (ETAAS). The results showed that Zn is mainly bound to 32 kDa (β-casein) protein (17.0 ± 2.0%) in UHT cow and 24 kDa (α-casein) protein (9.0 ± 0.6%) in raw sheep milk. This method provided quantitative information regarding Zn species present in the protein fractions of the milk samples. The accuracy was evaluated using certified reference material (whole milk powder, NIST 8435) with statistically equivalent concentrations (Student’s t-test) for total Zn and by addition and recovery experiments applied to measure Zn-protein. The recovered values were in the 92-110% range.

Identification of characteristic proteins of wheat varieties used to commercially produce dried noodles by electrophoresis and proteomics analysis

Characteristic proteins of wheat varieties used for commercial production of dried noodles were identified using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), acetic acid urea-polyacrylamide gel electrophoresis (AU-PAGE), two dimensional electrophoresis coupled with MALDI-TOF/TOF mass spectrometry analysis (2D-MS). Noodle textural properties prepared from the wheat varieties were also evaluated. The results of SDS-PAGE and texture evaluation of the noodles showed that some high molecular weight glutenin subunits (HMW-GSs), including subunits 5 + 10, 1, 7 + 8, 7 + 9, 7, 13 + 16, 20, 2 + 12, and 4 + 12 were possible desirable subunits of noodle wheat variety. The AU-PAGE analysis of gliadin extracted from the noodle wheat varieties demonstrated that five wheat varieties (SJ, HMT, YJ, DZ, and ZM) consisted of 1BL/1RS translocations. But in view of the noodle textural properties prepared from the five wheat varieties, we can conclude that desirable HMW-GSs existed in the wheat varieties can make up for the defects brought by 1BL/1RS translocations. Further identification of characteristic proteins of the wheat varieties by 2D-MS demonstrated that β-amylase, serpin, and α-amylase inhibitor, etc. were also potential characteristic proteins that affected the application of wheat variety. The study provided useful information for the selection and genetic breeding of wheat variety used to commercially produce dried noodles.

Mass Spectrometry-Based Identification of Urinary Antimicrobial Peptides in Dairy Cows.

Urine is considered one of the biological fluids in which antimicrobial peptides are secreted or expressed. Cow urine has not been investigated for the presence of these peptides using MALDI-TOF-MS. The aim of this study is to isolate, identify and assess the antimicrobial activity of urinary antimicrobial peptides from healthy normal cycling cows. We analyzed the urine sample using diafiltration, ion exchange chromatography, Reverse Phase High-Performance Liquid Chromatography (RP-HPLC), acid urea polyacrylamide gel electrophoresis (AU-PAGE) coupled with identification through Peptide Mass Fingerprinting (PMF) by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDITOF- MS). The in vitro antimicrobial effects of purified fractions were assessed using Radial Diffusion Assay (RDA) and microtitre broth dilution assay against Gram-positive and Gramnegative bacteria. Proteins corresponding to the peaks were identified using SWISSPROT protein database. This study revealed constitutive expression of β-Defensin-1 (DEFB1), β-Defensin-4A (DFB4A), Neutrophil Defensin-1 (DEF1), Neutrophil Defensin-3 (DEF3) in cow urine. The identified peptides are cationic antimicrobial peptides of the defensin family. The purified fractions exhibited antimicrobial effects in radial diffusion assay and MIC values in the range of 2.93-29.3 µM/L. This study concludes that cow urine, previously unexplored with regard to antimicrobial peptides, would be a promising source of highly potent AMPs and an effective alternative to the resistant antibiotics.

Utilization of natural stabilizer to prepare liposomal conjugate for the newly developed aptamer

Background: Aptamers are peptide or oligonucleotides that have the capacity to bind to a specific target molecule in human body. One of the newly developed aptamers named BAS aptamer. For targeting improvement of the aptamer, liposomal conjugate was adapted utilizing phosphatidylcholine as a natural lipid stabilizer in a comparison to synthetic one. Method: Two types of blank DSPE-PEG 2000-maleimide liposomes were prepared using two types of stabilizers; synthetic (Dipalmitoylphosphatidylcholine; DPPC) and natural lipids (phosphatidylcholine) then conjugated with BAS-aptamer through introducing thiol moiety. The liposomal conjugates then evaluated by H1-NMR and urea polyacrylamide gel electrophoresis and its efficacy on binding affinity, anticancer activity against MCF-7 breast cancer as well as its stability in plasma and buffers were estimated. Results: The binding affinity study revealed a significantly higher affinity of the naturally stabilized BAS aptamer conjugated liposomes towards drug receptor (SIRT1) than the synthetically stabilized one (KD= 38.5 ± 0.985 and 68± 0.967; respectively). The cytotoxic effect of the liposomal conjugates was higher than those of pure BAS aptamer and its thiol derivative in all concentrations used. The stability study in plasma showed that naturally stabilized liposomes remained stable for up to 6 days while the synthetically prepared liposomes showed partial degradation after 5 days and both gave higher stability than the pure BAS aptamer while the stability study in phosphate buffer at pH 7.4 and 5.5 revealed non-significant decline in the absorbance of the drug started after 6 days in its naturally stabilized conjugate and after 3 days (at pH5.5) its synthetically stabilized liposomal. Conclusion: Using natural stabilizer for liposomal conjugates gave better physical properties as well as significant binding affinity for the aptamer conjugated liposomes to the receptor (SIRT1) and higher cytotoxic effect as well as better stability in plasma and buffers.

Proteolysis in Irish farmhouse Camembert cheese during ripening.

Proteolysis in an Irish farmhouse Camembert cheese was studied during 10weeks of ripening. Urea-polyacrylamide gel electrophoresis of pH 4.6-insoluble fractions of cheese showed the degradation of caseins, initially due to the action of chymosin and plasmin and later due to Penicillium camemberti proteinases. Proteolytic specificities of Penicillium camemberti proteinases on the caseins in milk hydrolysates were determined and 64, 6, 28, and 2 cleavage sites were identified in αs1 -, αs2 -, β-, and κ-casein, respectively. Proteolysis in cheese was studied and peptides produced were determined and compared to the cleavage specificities of Penicillium camemberti proteinases. Regions most susceptible to proteolysis were 1-40, 79-114, and 168-199 in αs1 -casein; 42-79 and 97-116 in αs2 -casein; 40-57, 101-125, 143-189, and 165-209 in β-casein; and 31-81 and 124-137 in κ-casein. The present study describes in detail the proteolytic action of proteinases from Penicillium camemberti in Camembert cheese during ripening. PRACTICAL APPLICATIONS: Camembert cheese is a major international cheese variety, made in many countries around the world. The ripening of the cheese involves many biochemical changes and this study provides new information on peptides produced during ripening. Penicillium camemberti is an important mold used in the production of this type of cheese and detailed information is provided on the action of its enzymes on the caseins. Data reported in this study furthers the understanding of the ripening of Camembert cheese.

Cheese proteolysis and matrix disintegration during in vitro digestion

Abstract The proteolytic changes under in vitro digestive conditions were examined by urea-polyacrylamide gel electrophoresis, Kjeldahl determination of nitrogen, and fluorescamine analysis to evaluate the level of free amino terminal groups as an indicator of the degree of proteolysis. The protein and fat components of cheese digesta were visualized by confocal laser scanning microscopy. Cheese disintegration and subsequent release of proteins and polypeptides was quantified after each of three in vitro digestion phases: oral, gastric (pepsin, pH 2), and intestinal (pancreatic enzymes, pH 6.8). Most of the disintegration of the cheese protein matrix (˜75%), proteolysis, and protein release occurred during the intestinal digestion phase. Fat aggregate release from the cheese matrix mostly occurred in the gastric and intestinal digestion phases, and globules became more spherical after the gastric phase. Proteolysis took place primarily in the gastric and intestinal phases. These results show the importance of microstructure and the digestive environment on the release of cheese components.