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In recent years we have witnessed the emergence of organohalogen utilization in various chemical-based industries, particularly polymer-based, agricultural, and pharmaceutical sectors. Despite this, organohalogen compounds are actually very dangerous to the environment, as they are difficult to be naturally degraded and generally toxic to organisms. A green and biocompatible method to overcome this issue is by employing enzymes that could convert organohalogens into non-toxic compounds, such as the class of enzymes known as haloacid dehalogenases. To enhance the activity of haloacid dehalogenase isolated from local strains of Bacillus cereus IndB1, we have developed a recombinant expression system using pET-bcfd1 plasmid in E. coli BL21 (DE3) host cells. Following enzyme production, we also demonstrated a one-pot purification system for the expressed dehalogenase, harnessing the presence of His-tag in the recombinant clones. Purification was carried out using Ni-NTA affinity column chromatography, using imidazole eluent with a concentration gradient of 10 mM to 500 mM. The enzyme activity was tested against the monochloroacetic acid (MCA) substrate according to the Bergmann and Sanik method, and the protein content in the solution was measured using the Bradford method. The purity of the enzyme after one-pot purification was confirmed by SDS-PAGE analyses, showing a single band of 40 kDa in size. Remarkably, the purified haloacid dehalogenase specific activity was increased by 12-fold compared to its crude enzyme extract. Therefore, the expression and purification system developed in this study allow further exploration of dehalogenases from local strains as an efficient catalyst for MCA biodegradation.Keywords: recombinant expression, haloacid dehalogenase, monochloroacetic acid, enzyme purification

Toad skin secretions are sources of complex mixtures of bioactive compounds, such as proteins and peptides. Rhinella jimi species is a common toad in the Brazilian northeast, considered by only a few known studies. The experimental design was applied to optimize the protein extraction method from R. jimi parotoid gland secretions. The optimum condition was using 100 mmol L−1 Tris-HCl buffer pH 7.2 under vortexing for 5 min. The FTIR analysis combined with PCA revealed high-protein purity of the extracts, confirming the success of the proposed extraction method. The total protein concentration by the Bradford method was 102.4 and 66.5 mg g−1 on toad poisons from Teresina and Picos, respectively. The comparative proteomic analysis using HPLC-SEC-DAD and 1D SDS-PAGE revealed significant differences in protein abundance. HMW biomolecules showed greater abundance in toads from Teresina, while LMW protein species were more abundant in toads from Picos. The significant difference in amphibian proteome can be attributed to the edaphoclimatic conditions of their habitat. The cytotoxicity of the protein extract from Teresina was higher on the tumor cell lines 4T1 and CT26.WT. These new findings are fundamental for future studies the on identity and biological activity of biomolecules from this noble sample.

Extracellular vesicles (EVs) play essential roles in biological systems based on their ability to carry genetic and protein cargos, intercede in cellular communication and serve as vectors in intercellular transport. As such, EVs are species of increasing focus from the points of view of fundamental biochemistry, clinical diagnostics, and therapeutics delivery. Of particular interest are 30-200 nm EVs called exosomes, which have demonstrated high potential for use in diagnostic and targeted delivery applications. The ability to collect exosomes from patient biofluid samples would allow for comprehensive yet remote diagnoses to be performed. While several exosome isolation methods are in common use, they generally produce low recoveries, whose purities are compromised by concomitant inclusion of lipoproteins, host cell proteins, and protein aggregates. Those methods often work on lengthy timescales (multiple hours) and result in very low throughput. In this study, capillary-channeled polymer (C-CP) fiber micropipette tips were employed in a hydrophobic interaction chromatography (HIC) solid-phase extraction (SPE) workflow. Demonstrated is the isolation of exosomes from human urine, saliva, cervical mucus, serum, and goat milk matrices. This method allows for quick (

New support was fabricated to enhance the enzyme activity of cellulase following immobilization. Functionalized core-shell magnetic gold nanoparticles were prepared and characterized by X-ray diffraction (XRD), vibrating sample magnetometer (VSM), scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Cellulase enzyme was immobilized on support via covalent bonding. The successful binding of the enzyme was chemically confirmed by Fourier-transform infrared spectroscopy (FTIR). The binding efficiency was 84% determined by Bradford assay. Filter Paper Activity (FPase) method was used to measure the enzyme activity at different temperatures (35–75 °C) and pH (2–8). The immobilized cellulase maintained 73% of its initial catalytic activity after 9 h and its activity is 0.78 mmol.ml−1. The newly designed nano-system also enhanced the thermal stability of immobilized cellulase in comparison to free cellulase and facilitated its long term storage.

Metal ions like Cu2+ and Zn2+ have been shown to impact protein misfolding pathways in neurodegenerative proteinopathies like Alzheimer's and Parkinson's. Also, due to their strong interaction with Ubiquitin, they interfere in degradation of misfolded proteins by impairing the ubiquitin-proteasome system (UPS). In this work, we have studied the interaction of these metal ions with a small Ubiquitin like post-translation modifier SUMO1, which is known to work co-operatively with Ubiquitin to regulate UPS system. Between Cu2+ and Zn2+, the former binds more strongly with SUMO1 as determined using fluorescence spectroscopy. SUMO1 aggregates, forming trimer and higher oligomers in presence of Cu2+ ions which were characterized using gel electrophoresis, Bradford assay, and transmission electron microscopy. Chemical shift analysis using 15N/1H based NMR spectroscopy revealed that SUMO1 retains its structural fold in its trimeric state. Cu2+ induced paramagnetic quenching and Zn2+ induced chemical shift perturbation of 15N-1H cross-peaks were used to identify their respective binding sites in SUMO1. Binding sites so obtained were further validated with molecular dynamics studies. Our findings provide structural insights into the SUMO1-Cu2+/Zn2+ interaction, and its impact on aggregation of SUMO1 which might affect its ability to modify functions of target proteins.

Opiorphin secreted into the human saliva is an endogenous regulator pentapeptide (QRFSR) showing a pain suppressant effect similar to morphine. We report here a strategy of producing tandemly repeated multimers of opiorphin peptide efficiently by expressing in Esherichia coli cells. For improving the expression level and increasing the yield of pentapeptide, 32-mer tandem sequence of opiorphin was generated by using partially complementary primers inserted into pGEX-2T expression vector, following fused with glutathione transferase (GST) gene. Recombinant tandem multimer was easily captured by GST column with a yield of 9.2 mg/L, and then 32-mer opiorphin polypeptide of 28.49 kDa size was cleaved into monomers by CNBr and endoproteinase Asp-N. Opiorphin polypeptides having a molecular weight of about 693 Da and containing 5 amino acids in length were then generated. The amount of cleaved and purified recombinant opiorphin monomers was calculated as 6.2 mg/L by the Bradford assay. Protein expression and cleavage of recombinant polypeptide were confirmed by SDS-PAGE and TLC. Results from this study clearly open a door to the mass production of the opiorphin peptide to use for laboratory research and industrial purposes. The overall gene construction and the direct cloning to an expression vector strategy using partially complementary primers with cleavable linker peptides could be applicable to many small peptides and their analogs for pharmaceutical and clinical application.

An engineered Pichia pastoris GS115 with a FIP-glu gene was mutated using ultraviolet (UV) radiation, and a high-throughput screening method was established for screening of high-yield strains. Meanwhile, a preliminary study was conducted to determine the bioactivity of the rFIP-glu. Based on OD600 value and the mortality of engineered P. pastoris GS115, the best UV irradiation time was determined. Bradford method and SDS-PAGE method were employed to analyze the concentration and yield of rFIP-glu. Melanoma B16 cells were employed to evaluate the biological activities of rFIP-glu in vitro. Results showed that the protein yield of the best mutant #4-336 screened from 3680 mutant strains increased from 242 to 469 μg ml-1 . In vitro assays of biological activity indicated that rFIP-glu had significant toxicity and possessed the ability to affect melanin content and enhance tyrosinase activity in B16 cells. In conclusion, an effective high-throughput screening approach was established for screening mutant strains. The screened mutant possesses a good ability to enhance the production of rFIP-glu, and recombinant proteins display a better biological activity on melanoma B16 cells. The engineered P. pastoris mutant seems promising as a potential source for industrial production of rFIP-glu and should be a candidate industrial strain for further study.

PDMS based multi-channel microfluidic chip was designed and fabricated in a simple approach using readily available tools. UV-initiated in situ polymerization of poly(2-hydroxy ethyl methacrylate-co-di(ethylene glycol) diacrylate-co-N,N'-diallyl L-tartardiamide) in an eppendorf tube was achieved within 40 min. This polymerization process was successfully translated to a microfluidic chip format without any further modifications. Iminodiacetic acid was successfully immobilized on aldehyde functional monoliths via Schiff base reaction and confirmed by FT-IR spectroscopy. Four transition metal-ions (Co (II), Zn (II), Ni (II), and Cu (II)) were chelated individually on four IDA-monolith microfluidic chips. The conjoint metal-ion monolith microfluidic chip has displayed high permeability (9.40 × 10-13 m2 ) and a porosity of 32.8%. This affinity microfluidic chip has pre-fractioned four human plasma proteins (fibrinogen, immunoglobulin, transferrin and human serum albumin) based on their surface exposed histidine surface topography. A protein recovery of ∼95% (Bradford assay data) was achieved. The multi-monolith microchip can be reusable even after 3 protein adsorption-desorption cycles. This article is protected by copyright. All rights reserved.

One of the neuropathological hallmarks of Alzheimer's disease (AD)—causing neurodegeneration and consequent memory deterioration, and eventually, cognitive decline—is amyloid-β (Aβ) aggregation forming amyloid plaques. Our previous study showed the potential of a tocotrienol-rich fraction—a mixture of naturally occurring of vitamin E analogs—to inhibit Aβ aggregation and restore cognitive function in an AD mouse model. The current study examined the effect of three vitamin E analogs—α-tocopherol (α-TOC), α-tocotrienol (α-T3), and γ-tocotrienol (γ-T3)—on Aβ aggregation, disaggregation, and oligomerization in vitro. Thioflavin T (ThT) assay showed α-T3 reduced Aβ aggregation at 10 μM concentration. Furthermore, both α-T3 and γ-T3 demonstrated Aβ disaggregation, as shown by the reduction of ThT fluorescence. However, α-TOC showed no significant effect. We confirmed the results for ThT assays with scanning electron microscopy imaging. Further investigation in photo-induced cross-linking of unmodified protein assay indicated a reduction in Aβ oligomerization by γ-T3. The present study thus revealed the individual effect of each tocotrienol analog in reducing Aβ aggregation and oligomerization as well as disaggregating preformed fibrils., Highlights • Tocotrienols (T3) reduce amyloid-β (Aβ) aggregation and disaggregate fibrils. • γ-T3 is the most effective vitamin E analog to reduce Aβ oligomerization. • Structure conformation of vitamin E may influence the effect on Aβ peptides. • T3 is a promising therapeutic compound to treat Alzheimer's disease.

Protein-nanoparticle (NP) interaction, which inevitably form protein corona (PC), has been the subject of much debate about its role in modern biomedical research. In this regard, PC associated with two different NPs viz., magneto-fluorescent (MF) and chitosan coated MF (CMF) NPs were thoroughly investigated, to analyze the effect of polymer coating on protein adsorption. Bradford assay, along with the spectroscopic and microscopic studies suggested increase in adsorbed protein quantity, though the results varied significantly on moving from bare to polymeric coating and in vitro to ex vivo conditions. Interestingly, polymer coated NPs showed increased protein adsorption and induce minimal changes in protein structural integrity under the same conditions. We predict that the changes in secondary structure of primary corona determine the overall signature of surface binding proteins in PC. Our findings suggested that rapid diagnosis of immunoglobulins is possible using the concept of protein corona formation ex vivo.

Keratin was extracted from different segments of disposable waste chicken feathers (CF) including the whole feathers, calamus/rachis (β-sheet) and barbs/barbules (α-helix), using sodium sulfide and l -cysteine. The yield of extracted keratin from sodium sulfide and l -cysteine was ˜88% and ˜66% respectively. The mass ratio of feathers to reducing agent was 1:20 and the reaction temperature was 40 °C for 6 h. Concentration of keratin extracted by each method was measured using the Bradford assay. The protein extracted from each feather section was characterised using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, vibrational spectroscopy including FTIR and Raman, nuclear magnetic resonance, and thermogravimetry. These results confirmed the keratin structures after each extraction methods. The study showed that α-helix and s-sheet based keratin could be extracted from CF using sodium sulfide and l -cysteine with high yields. This is the first report of CF keratin extraction using l -cysteine.

The novel antimicrobial gene Hirudomacin (Hmc), with a 249-bp cDNA, encodes a mature protein of 61 amino acids and a 22-amino acid signal peptide. Hmc exhibits the highest similarity, at 90.1%, with macin family members found in the salivary gland of the leech Hirudo nipponica Whitman. A mature Hmc protein concentration of 219 μg/mL was detected using the Bradford method. The mature Hmc protein is 6862.82 Da and contains 8 cysteine residues. Antimicrobial assays showed a minimum bactericidal concentration and 50% lethal dose of 1.56 μg/mL and 0.78 μg/mL, respectively, for Staphylococcus aureus and 0.39 μg/mL and 0.195 μg/mL, respectively, for Bacillus subtilis. Transmission electron microscopy revealed membrane integrity disruption in S. aureus and B. subtilis, which resulted in bacterial lysis. The level of Hmc mRNA in the salivary gland during three blood meal stages indicated a remarkable trend of increase (P .05), and western blotting demonstrated that among the three blood meal stages, expression of the mature Hmc protein was highest in both the salivary gland and intestine at the fed stage (P .05). Immunofluorescence further showed the mature Hmc protein to be localized outside the cell nucleus, with the signal intensity in the salivary gland peaking at the fed stage (P .05). In conclusion, the mature Hmc protein exhibits broad-spectrum antimicrobial effects against gram-positive and gram-negative bacteria, and a blood meal upregulates Hmc gene and protein expression in H. nipponica.

The Bradford assay is one of the most commonly used methods for protein quantification. However, in proteomic research, the lysis buffer generally used for dissolving proteins can cause some interference to the assay. The dye reagent of classical Bradford assay contains 8.50% (w/v) phosphoric acid, which is an important factor relating to the color yield of the assay. In this study, the phosphoric acid content in dye reagent was increased to 9.35% (w/v), 10.20% (w/v), and 11.05% (w/v) to evaluate the changes of interference and the effects of lysis buffer on the interaction between proteins and dye reagent. Results show that lysis buffer not only causes background interference but also affects the protein-dye chromogenic process. Analysis of different components in the lysis buffer showed that carrier ampholyte is the main factor that introduces interference to the Bradford assay. Detergents are well-known interfering compounds in the Bradford assay, but CHAPS and octyl b-D-glucopyranoside only cause slight interference. When the amount of phosphoric acid was increased from 8.50%(w/v) to 10.20% (w/v), the sensitivity of the Bradford assay to proteins in lysis buffer was increased, and the interference delivered by lysis buffer was considerably reduced.

Graphical abstract, Luciferase-based reporter assays are one of the most common cell-based screening formats for drug discovery, and simultaneous evaluation of the cytotoxic effect of test compounds is of great value in reducing false-positives. Here we share a multiplex assay protocol that allows sequential measurement of cell viability (cell number) and luciferase activity of the same sample in a multi-well-plate format. The viability assay employs a fluorogenic esterase substrate, CytoRed. • This protocol allows sequential measurement of endogenous esterase activity (as a surrogate for cell number) and then luciferase activity in a single sample. • The protocol eliminates the need for parallel viability assay or protein assay using separate aliquots of the lysate. • This protocol is especially useful for assays with cells stably expressing a luciferase construct, for which co-transfection of another reporter gene is not a viable option.

Recombinant flounder growth hormone was overproduced in E. coli by using codon optimized synthetic gene and optimized expression conditions for high level production. The gene was cloned into PET-28a expression vector and transformed into E. coli BL21 (DE3). Induction at lower temperature, lower IPTG concentrations and richer growth media during expression resulted in increased expression level. The protein expression profile was analyzed by SDS-PAGE, the authenticity was confirmed by western blotting and the concentration was determined by Bradford assay. In addition, several attempts were made to produce soluble product and all resulted in insoluble product. The overexpressed protein was efficiently purified from inclusion bodies by moderate speed centrifugation after cell lysis. Among the solubilization buffers examined, buffer with 1% N-lauroylsarcosine in the presence of reducing agent DTT at alkaline pH resulted in efficient solubilization and recovery. The denaturant was removed by filtration and dialysis. The amount of the growth hormone recovered was significantly higher than previous reports that expressed native growth hormone genes in E. coli. The methodology adapted in this study, can be used to produce flounder growth hormone at large scale level so that it can be used in aquaculture. This approach may also apply to other proteins if high level expression and efficient purification is sought in E. coli. Keywords: Expression, Purification, Recombinant, Growth hormone

The objective of the present study was to evaluate different methodologies for peptide quantification in the supernatant of chitosan nanoparticles by removing the unliked polymer in the suspension. The ionic gelation method was used to prepare the chitosan nanoparticles encapsulating a 5.3 kDa peptide. Three different methodologies for the processment of the solutions were compared before subjecting the samples to the Bradford protocol or Qubit® kit for protein detection. For the quantification, it was necessary to create a standard peptide curve using different peptide concentrations. The suitable standard curve would be one in which the peptide was diluted in the empty chitosan supernatant (obtained after nanoparticles centrifugation) or in the filtrate of the empty chitosan supernatant. Our results indicated the safest methodology tested for peptide quantification in the supernatant chitosan nanoparticles was filtering the solution before subjecting the sample to the Bradford assay.

Replacing synthetic polymers with renewable alternatives is a critical challenge for the packaging industry. This research investigated the use of leaf-based proteins as a sustainable co-binder in the coating formulations for paper-based packaging and other applications. Protein isolates from tobacco leaf and alfalfa concentrates were characterized using the Pierce protein assay, Kjeldahl nitrogen, and gel electrophoresis. The proteins were tested as co-binders in a typical latex-based paper coating formulation. The rheology and water retention properties of the wet coating and the surface, optical, structural, and strength properties of coated papers were measured. The coating performance was affected by the purity, solubility, and molecular weight of the tobacco protein and exhibited a shear-thinning behavior with lower water retention than soy protein. Analysis by scanning electron microscopy and time of flight secondary ion mass spectroscopy on the dried coating layer containing tobacco protein showed enhanced porosity (advantageous for package glueability) relative to the control latex coating. The tobacco protein offers adequate coverage and coating pigment distribution, indicating that this protein can be a suitable option in coatings for packaging applications.

Proteinuria is considered indicative of kidney damage that can be related to various adverse outcomes. Nowadays, there is a huge demand for routine urine screening methods to assess health risks in clinical setting without expensive procedures and long pretreatment of the sample. To address this issue, a polydopamine-based colorimetric assay to determine urinary albumin concentration in real samples is proposed here. The core of this approach relies on the established competitive adsorption of polydopamine film and human serum albumin onto the polystyrene surface of ELISA plates. Herein, we investigated the influence of temperature and the Tris-HCl buffer concentration on the polydopamine film growth. The absorbance of polydopamine film, after 24 h at 25 °C, decreases with the increase of HSA concentration, allowing the selective detection of HSA down to 0.036 ± 0.001 g L−1 in untreated urine. This simple and low-cost bioanalytical assay exhibited very good reproducibility, %CVmean = 2 in human urine, and was superior in terms of analytical performances to some standard methods available on the market, especially in comparison to the Bradford assay, for early screening and assessment of kidney damage.

Aims:: We aim to evaluate the potential application of amine reactive fluorogenic reagents for estimating enzymatic proteolysis. Background:: Proteolytic enzymes play important roles in regulating many physiological processes in living organisms. Objectives:: Assessment of protein degradation by using reagents for protein assay techniques. Methods:: We have assayed samples at the start and after 30-60 minutes incubation with trypsin by Chromeo P503 (Py 1 pyrylium compound) and CBQCA (3-(4-carboxybenzoyl) quinoline-2-carboxaldehyde) as amine reactive reagents and NanoOrange as non-amine reactive dye. Results:: All BSA prepared samples with trypsin have shown significantly higher fluorescence intensity (FI) versus controls (which reflects proteolysis) when assayed by Chromeo P503 (Py 1 pyrylium compound) and CBQCA (3-(4-carboxybenzoyl) quinoline-2-carboxaldehyde) as amine reactive reagents. However, same samples assayed with NanoOrange as non-amine reactive reagent did not show any significant variation between samples containing trypsin and controls. Conclusion:: These results are confirming reliability of highly sensitive protein assays utilizing amine reactive fluorogenic reagents for general estimation of proteolysis.

Chemiluminescent biosensors have been developed and broadly applied to mammalian cell systems for studying intracellular signaling networks. For bacteria, biosensors have largely relied on fluorescence-based systems for quantitating signaling molecules, but these designs can encounter issues in complex environments due to their reliance on external illumination. In order to circumvent these issues, we designed the first ratiometric chemiluminescent biosensors for studying a key bacterial second messenger, cyclic di-GMP. We have shown recently that these biosensors function both in vitro and in vivo for detecting changes in cyclic di-GMP levels. In this chapter, we present a practical and broadly applicable method for high-throughput quantitation of cyclic di-GMP in bacterial cell extracts using the high affinity biosensor tVYN-TmΔ that could serve as the "Bradford assay" equivalent for this bacterial signaling molecule.

To investigate the application of short elastin-like polypeptides (ELPs) in the purification of bioactive proteins, short hydrophobic ELP[I](n) (n = 30, 40, 50) tags were constructed. Both the ELP[I](n) tags and the ELP[I](n)–Trx fusion proteins could be stably expressed in Escherichia coli and purified by inverse transition cycling, respectively. Total protein concentrations determined by BCA protein assay showed that the yield of the fusion proteins decreased with increasing ELP length. Measurements of the inverse transition temperature (T(t)) of the ELP[I](n)–Trx under different salts or PEG8000 concentrations showed decreased T(t) upon elevated concentrations; while, all the T(t)s were suitable for generating proteins from 4 to 37.5 ºC. Furthermore, to identify a linker peptide for bioactive protein production without the need to remove the ELP[I](n) tag, the activity of eGFP protein fused with ELP[I](30) tag by either a poly-N or a G4S linker was quantified using a fluorescence spectrophotometer. The results indicated that the ELP[I](30)–eGFP fusion proteins with the poly-N linker showed higher fluorescence levels than those with the G4S linker. Our results demonstrated that short ELP[I](n) tags with low T(t) were useful in protein expression and purification, and poly-N linker played the key role in producing bioactive proteins without the need to remove the ELPs.

Production of anti-sperm antibody (ASA) often suffers from autoimmune reaction against sperms in human infertility. The antibodies are measured in both blood and seminal plasma of males. Here, we reported a simple protein biochip methodology that takes advantage of a functionalized self-assembled monolayer modified by N-hydroxysuccinimide (NHS) and enables identification of anti-sperm antibody in Chinese male infertility. To validate this biochip platform, we immobilized purified sperm protein on the biochip surface and tested a variety of parameters in quality controls for the protein assay, respectively. Then, we analyzed serum samples from 368 patients with infertility and 116 healthy donors by means of this biochip simultaneously. We found that positive rate of serum ASA was 20.92% (77/368) in the cases and 1.72% (2/116) in the controls, respectively. Furthermore, we further corroborated the biochip assay in comparison with ELISA method. We found that both methods were compatible for the detection of serum ASA in the patients. In addition, a follow-up study for natural conception in ASA-positive and ASA-negative patients was conducted. The result showed a significant correlation between serum ASA expression and natural pregnancy rate 6.5% in ASA-positive patients while 18.9% in ASA-negative patients, indicating the potential roles of ASA in naturally reproductive processes.

Accurate determination of residual protein content in hydroxypropyl chitin (HPCH), a temperature-sensitive chitin derivative developed recently, is of great importance for biomedical applications. Coomassie brilliant blue (Bradford) method, Lowry method and Bicinchoninic acid (BCA) method were investigated to estimate the content of residual protein in HPCH. It was found that both Bradford and Lowry methods are not suitable for the protein assay in the HPCH sample. The BCA method could be used to determine protein content in HPCH solutions at low concentrations with good accuracy and precision and reasonable limit of detection and limit of quantitation for medical applications.

Sensitive and specific detection of protein is of great significance for early diagnosis and prognosis of many diseases. However, great challenges remain unsolved including relative low sensitivity, high cost, long testing time, complicated instrument and laborious operation. To improve the performance of protein detection methods, development of fine reaction interface for recognition and signal amplification is of great importance. In this work, we construct a novel mode of DNA assembly at electrode interface based on a tripodal surface anchor and an electrochemical aptasensor for protein assay is developed. The orientation of the immobilized DNA is optimized, which promises the efficiency of protein recognition. In addition, hybridization chain reaction is employed for further signal amplification. Therefore, this detection method shows high sensitivity with excellent specificity. The strategy can be universally applicable by simply modifying the sequences of used DNA probes.

Human growth hormone (hGH) is synthesized by the anterior pituitary gland and promotes cell proliferation and growth. This protein has been authorized to use for the treatment of various human growth disorders and until recently, substantial efforts have been made to upgrade the previous introduced strategies. Due to the small size of hGH and absence of posttranslational modifications, Escherichia coli is the ideal host for hGH production. In the present work, we employed a previously established cleavable self-aggregating tag (cSAT) for the expression and purification of hGH in BL21 (DE3) strain of E. coli to evaluate its effectiveness. The tag is composed of a self-cleavable intein and a self-assembling peptide ELK16 (Mxe GyrA intein-ELK16). At the first step, an active insoluble aggregate of the recombinant hGH-Mxe GyrA intein-ELK16 fusion protein was expressed through an efficient T7 based expression system and then purified with a simple centrifugation. The thiol reagent dithiothreitol (DTT) was then added to induce the intein-mediated cleavage and as a result the peptide released into the soluble fraction. Afterward, the hGH production was determined by SDS-PAGE and then the final concentration of released hGH was measured by the Bradford assay (4.96 mg/ml). Furthermore, the bioactivity of purified hGHs was confirmed by calculating its growth-stimulating effect using Nb2 cell line proliferation assay. All in all, the current study offers a straightforward and fast procedure for the production of pure and bioactive hGH in E. coli.

Small molecules are not only intermediates of metabolism, but also play important roles in signaling and in controlling cellular metabolism, growth, and development. Although a few systematic studies have been conducted, the true extent of protein-small molecule interactions in biological systems remains unknown. PROtein-metabolite interactions using size separation (PROMIS) is a method for studying protein-small molecule interactions in a non-targeted, proteome- and metabolome-wide manner. This approach uses size-exclusion chromatography followed by proteomics and metabolomics liquid chromatography-mass spectrometry analysis of the collected fractions. Assuming that small molecules bound to proteins would co-fractionate together, we found numerous small molecules co-eluting with proteins, strongly suggesting the formation of stable complexes. Using PROMIS, we identified known small molecule-protein complexes, such as between enzymes and cofactors, and also found novel interactions. © 2019 The Authors. Basic Protocol 1: Preparation of native cell lysate from plant material Support Protocol: Bradford assay to determine protein concentration Basic Protocol 2: Separation of molecular complexes using size-exclusion chromatography Basic Protocol 3: Simultaneous extraction of proteins and metabolites using single-step extraction protocol Basic Protocol 4: Metabolomics analysis Basic Protocol 5: Proteomics analysis.

There is a need to adapt cell bioassays to 384-well and 1536-well formats instead of the traditional 96-well format as high-throughput screening (HTS) demands increase. However, the sensitivity and performance of the bioassay must be re-verified in these higher micro-well plates, and verification of cell health must also be HT (high-throughput). We have adapted two commonly used human breast luciferase transactivation cell bioassays, the recently re-named estrogen agonist/antagonist screening VM7Luc4E2 cell bioassay (previously designated BG1Luc4E2) and the androgen/glucocorticoid screening MDA-kb2 cell bioassay, to 384-well formats for HTS of endocrine-active substances (EASs). This cost-saving adaptation includes a fast, accurate, and easy measurement of protein amount in each well via the fluorescamine assay with which to normalize luciferase activity of cell lysates without requiring any transfer of the cell lysates. Here we demonstrate that by accounting for protein amount in the cell lysates, antagonistic agents can easily be distinguished from cytotoxic agents in the MDA-kb2 and VM7Luc4E2 cell bioassays. Additionally, we demonstrate via the fluorescamine assay improved interpretation of luciferase activity in wells along the edge of the plate (the so-called "edge effect"), thereby increasing usable wells to the entire plate, not just interior wells.

A new modular nanoswitch was described for versatile, rapid (within 1 h), homogeneous, and sensitive protein detection. The system employs two hairpins (HP1 and HP2) that can be reciprocally recognized through the apical loop-loop interaction. HP2 possesses a conformation-switching stem-loop structure, with appended single-stranded tails on each end, which can hybridize with the recognition-element-conjugated DNA strands to construct a protein-responsive HP2 scaffold. It works according to a simple mix-and-detect assay format, with the first formation of a kissing complex between HP1 and HP2 scaffolds for fluorescence quenching, and then cascade propagation from steric strain through protein binding to the dissociation of the kissing complex for fluorescence recovery. The detection universality of such a modular nanoswitch was demonstrated by using three multivalent proteins, including anti-digoxigenin (Anti-Dig) antibody, streptavidin, and thrombin, with detection limits of 0.33, 0.17, and 0.5 nm, respectively.

Detection of molecular biomarkers with high specificity and sensitivity from biological samples requires both sophisticated sample preparation and subsequent analysis. These tasks are often carried out on separate platforms which increases required sample volumes and the risk of errors, sample loss, and contamination. Here, we present an optofluidic platform which combines an optical detection section with single nucleic acid strand sensitivity, and a sample processing unit capable of on-chip, specific extraction and labeling of nucleic acid and protein targets in complex biological matrices. First, on-chip labeling and detection of individual lambda DNA molecules down to concentrations of 8 fM is demonstrated. Subsequently, we demonstrate the simultaneous capture, fluorescence tagging and detection of both Zika specific nucleic acid and NS-1 protein targets in both buffer and human serum. We show that the dual DNA and protein assay allows for successful differentiation and diagnosis of Zika against cross-reacting species like dengue.

Deep-ultraviolet laser ablation with a pulsed 193 nm ArF excimer laser was used to remove localized regions from tissue sections from which proteins were extracted for spatially resolved proteomic analysis by liquid chromatography tandem mass spectrometry (LC-MS/MS). The ability to capture intact proteins by ablation at 193 nm wavelength was verified by matrix-assisted laser desorption ionization (MALDI) of the protein standard bovine serum albumin (BSA), which showed that BSA was ablated and captured without fragmentation. A Bradford assay of the ablated and captured proteins indicated 90% efficiency for transfer of the intact protein at a laser fluence of 3 kJ/m2. Rat brain tissue sections mounted on quartz microscope slides and ablated in transmission mode yielded 2 μg protein per mm2 as quantified by the Bradford assay. Tissue areas ranging from 0.06 mm2 to 1 mm2 were ablated and the ejected material was collected for proteomic analysis. Extracted proteins were digested and the resulting peptides were analyzed by LC-MS/MS. The proteins extracted from the ablated areas were identified and the average number of identified proteins ranged from 85 in the 0.06 mm2 area to 2400 in the 1 mm2 area of a 50 μm thick tissue . In comparison to infrared laser ablation of equivalent sampled areas, both the protein mass and number of proteins identified using DUV laser ablation sampling were approximately four times larger.

Anacystis nidulans is a small, rod-shaped, unicellular, colonial, obligatory phototrophic microalga isolated from Sambhar Lake, Jaipur (Rajasthan). To find out the best inorganic composition cultures were grown in five different defined inorganic medium such as Modified BG-11 medium (pH 7.31), BG-11 medium (7.1), CHU-10 (pH 7.65), Zarrouk’s medium (pH 10.2) and Kratz & Myer medium (pH 9.5) and kept at the temperature of 25 ± 2°C, illuminated with white fluorescent lamps at a light intensity of 2.5 Klux with 12:12 hours light/dark photoperiod in departmental laboratory. Protein content is determined by Bradford assay and qualitatively by SDS-PAGE. Protein expression levels were determined through densitometry. Highest protein and amino acid content were obtained in Modified BG-11 medium as compared to other medium. Two polypeptides of 54.3 and 56.2 kDa were uniquely observed, but the genotype of 35.8 kDa polypeptide was completely degraded under Modified BG-11 inorganic formulation. 35.8, 54.3, 56.2 and 61.8 kDa polypeptides were completely degraded in Zarrouk’s as well as Kratz and Myer medium. The expression of some polypeptides of 14.0, 34.1, 42.3, 45.9, 49.5 and 75.0 kDa were greatly reduced and expressed only 1mm level in Zarrouk’s and Kratz and Myer medium. Quantity of free amino acids maximum was in Modified BG-11 medium and minimum was in the Kratz and Myer Medium. Total 17amino acids were observed in the HPLC chromatogram. No detectable amount of asparagine, glutamine and tryptophan were found throughout the course of the algal life cycle

In the present study, the disruptive effects of epigallocatechin-3-gallate (EGCG) and A-type dimeric epigallocatechin-3-gallate (A-type EGCG dimer) on the preformed bovine insulin amyloid fibrils were studied by several biophysical methods including thioflavin-T (ThT) fluorescence assay, 1-anilinonaphthalene-8-sulfonic (ANS) fluorescence assay, Congo red (CR) binding assay, dynamic light scattering (DLS), transmission electron microscopy (TEM), Gel electrophoresis (SDS-PAGE) and Bradford assay. Our results demonstrated that A-type EGCG dimer showed significantly more potential disaggregative effects on the bovine insulin amyloid fibrils than EGCG. A-type EGCG dimer could not only dramatically promote the disaggregation of the preformed bovine insulin amyloid fibrils, but also restructure the amyloid fibrils into amorphous aggregates. While, EGCG could only shorten and thin the fibrils, but induce no small amorphous aggregates. Our present results provided additional evidence for the more potent disaggregation effects of dimeric polyphenols than monomeric polyphenols and suggested that A-type EGCG dimer seems to have potential application as an excellent anti-amyloidogenic agent.

This study aimed to provide a novel and highly sensitive protein assay based on the biuret reaction and using chromeazurol B, a metal chelate compound. The method consists of two reagents and an automated analyzer. First, a complex of copper and protein (biuret reaction) is formed. Second, a chelating reagent containing chromeazurol B forms a three-dimensional complex of protein, copper, and chromeazurol B at neutral pH, resulting in highly sensitive coloration. The intra-assay (n = 20) variation for the three levels was 3.54 % or lower at each concentration. Each response with α, β-, and γ-globulin was 103.8 % and 104.3 %, respectively, against albumin. The molar absorption coefficient (e) of the present method was 2.5 × 105 m2/mol against human albumin, higher than that of the commercially available Lowry method (e = 8.7 × 104 m2/mol), which is based on the same principle. The correlation test for the pyrogallol method with 30 urine samples showed good performance (r = 0.961). The method described here (the Biuret-based CAB method) is a more sensitive and rapid assay than the Lowry method, and it may also be applied to biological samples because of its similar reactivity towards various proteins.

Culture broth of Ganoderma lucidum was determined for antioxidant, antibacterial and α-amylase inhibitory activities. The culture broth contained protein as determined by Bradford method equaled to 0.2 mg/ml and total phenol content as 0.078 mg GAE/mg protein (0.557 mg GAE/g extract). It exhibited radicals scavenging activities against ABTS+· and DPPH· radicals with a half maximal inhibitory concentration (IC50) less than 1.70 ± 0.02 and 2.28 ± 0.02 µg protein/ml, respectively and reducing power equaled to 4.38 ± 0.02 µmol Trolox/µg protein as investigated by ferric ion reducing antioxidant power method. The culture broth experimented into two approached; (1) treated with pronase and (2) filtered through a membrane with 10 kDa molecular weight cut-off (MWCO). The pronase-treated culture broth exhibited insignificant lower antioxidant activities, but the retained culture broth 10 kDa MWCO resulted in significant decrease in antioxidant activities suggesting that the small proteins might play the key role in antioxidant activity. The culture broth could protect DNA damage from hydroxyl radicals (·OH) generated by Fenton’s reaction. This culture broth showed antibacterial activity towards pathogenic strains Staphylococcus epidermidis and Pseudomonas aeruginosa and also had an interesting α-amylase inhibitory activity. This study suggested that apart from the fruiting bodies and the mycelial of G. lucidum, its culture broth also had potential applications as a value-added ingredient in the product such as in cosmetics and in nutraceuticals.

The Bradford reagent, comprised of the Coomassie Brilliant Blue G-250 dye, methanol, and phosphoric acid, has been traditionally used for quantifying proteins. Use of this reagent in the Bradford assay relies on the binding of the Coomassie Blue G-250 dye to proteins. However, the ability of the dye to react with a small group of amino acids (arginine, histidine, lysine, phenylalanine, tyrosine, and tryptophan) makes it a viable chemical assay for fingerprint analysis in order to identify the biological sex of the fingerprint originator. It is recognized that the identification of biological sex has been readily accomplished using two other methods; however, both of those systems are reliant upon a large group of amino acids, 23 to be precise. The Bradford assay, described here, was developed specifically to aid in the transition from targeting large groups of amino acids, as demonstrated in the previous studies, to targeting only a single amino acid without compromising the intensity of the response and/or the ability to differentiate between two attributes. In this work, we aim to differentiate between female fingerprints and male fingerprints.

The effects of antibacterial compounds produced by Saccharomyces cerevisiae in Koumiss on pathogenic Escherichia coli 0 8 and its cell surface characteristics were investigated. S. cerevisiae isolated from Koumiss produced antibacterial compounds which were active against pathogenic E. coli O 8 as determined by the Oxford cup method. The aqueous phases from S. cerevisiae at pH=2.0 (S2) and pH=8.0 (S8) were extracted and tested, respectively. The organic acids of S2 and S8 were determined by high performance liquid chromatography (HPLC), and the concentrations of killer toxins were determined by enhanced bicinchoninic acid (BCA) Protein Assay Kit. The minimum inhibition concentration (MIC) and the minimum bactericidal concentration (MBC) of S2 and S8 on E. coli O 8 were determined by the broth microdilution method. The effects of S2 and S8 on the growth curve of E. coli O 8 were determined by turbidimetry, and the hydrophobicities of E. coli O 8 cell surface were determined using the microbial adhesion to solvents method, the permeation of E. coli O 8 cell membrane were determined by the o-nitrophenyl-β-D-galactoside (ONPG) method. Aqueous phases at pH 2.0 and 8.0 had larger inhibition zones and then S2 and S8 were obtained by freeze-drying. The main component in S2 was citric acid and it was propanoic acid in S8. Other organic acids and killer toxins were also present. Both the MICs of S2 and S8 on E. coli O 8 were 0.025 g mL −1 , the MBCs were 0.100 and 0.200 g mL −1 , respectively. The normal growth curve of E. coli O 8 was S-shaped, however, it changed after addition of S2 and S8. E. coli O 8 was the basic character, and had a relatively hydrophilic surface. The hydrophobicity of E. coli O 8 cell surface and the permeation of E. coli O 8 cell membrane were increased after adding S2 and S8. The present study showed that S2 and S8 inhibit the growth of pathogenic E. coli O 8 and influence its cell surface characteristics.

The aim of this manuscript was to study the application of a new method of protein quantification in Candida antarctica lipase B commercial solutions. Error sources associated to the traditional Bradford technique were demonstrated. Eight biocatalysts based on C. antarctica lipase B (CALB) immobilized onto magnetite nanoparticles were used. Magnetite nanoparticles were coated with chitosan (CHIT) and modified with glutaraldehyde (GLUT) and aminopropyltriethoxysilane (APTS). Later, CALB was adsorbed on the modified support. The proposed novel protein quantification method included the determination of sulfur (from protein in CALB solution) by means of Atomic Emission by Inductive Coupling Plasma (AE-ICP). Four different protocols were applied combining AE-ICP and classical Bradford assays, besides Carbon, Hydrogen and Nitrogen (CHN) analysis. The calculated error in protein content using the "classic" Bradford method with bovine serum albumin as standard ranged from 400 to 1200% when protein in CALB solution was quantified. These errors were calculated considering as "true protein content values" the results of the amount of immobilized protein obtained with the improved method. The optimum quantification procedure involved the combination of Bradford method, ICP and CHN analysis.

Background and Purpose: Allergy is an undesired immune response to non-pathogenic agents. However, some opportunistic microorganisms such as fungi can also cause allergy. Among those fungi, hyphae form of Aspergillus strains including A. fumigatus, A. flavus, and A. niger could be mentioned. In this study, we aimed to separate allergic proteins from Aspergillus strains and determine their identity. Materials and Methods: Standard species of Aspergillus strains were cultivated in optimized conditions and the mycelium was separated by centrifugation. The fungal cells were lysed through physical methods such as freezethawing and grinding to prepare a suitable protein extract. The protein concentration was measured by Bradford method and the electrophoretic pattern of the extract was determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The proteins were fractionated by ammonium sulfate precipitation and anion exchange chromatography using fast protein liquid chromatography (FPLC) system. The IgE immunoreactivity of the sensitized patients and controls was studied using the fractionated proteins by enzyme-linked immunosorbent assay (ELISA). Following SDS-PAGE, proteins were electrotransferred onto polyvinylidene difluoride (PVDF) membranes and the strips were blotted with allergic patients' and controls' sera. The immunoreactive bands were excised from colloidal coomassie-stained SDS-PAGE gels and studied by mass spectroscopy methods. Results: Among the studied species, A. fumigatus showed stronger IgE reactivity and more IgE reactive protein bands than others did. The proteins with higher molecular weights showed stronger immunoreactivity in Western blotting. Receiver operating characteristic curve analysis demonstrated a correlation between the results of the applied ELISA methods. One of the most prominent IgE reactive proteins was confirmed to be 45 kDa mycelia catalase. Conclusion: Our findings confirmed that high molecular weight proteins might play a major role in allergy and IgE reactivity to Aspergillus species. Moreover, the results showed that precipitation and chromatographic methods are applicable for fractionation of fungal proteins such as mycelial catalase. &nbsp

Ethnopharmacological relevance Panax quinquefolius L. has been used as a proverbial tonic in oriental countries for hundreds of years. It is used as a traditional medicinal herb to nourish vitality. Aim of the study The purpose of our study was to inquiry the activation effects on murine peritoneal macrophages of a novel protein separated from the roots of Panax quinquefolius L. Materials and methods In our work, a novel protein of the roots of American ginseng (AGNP) was separated and purified from the roots of Panax quinquefolius L. The characteristic was investigated with SDS-PAGE, high pressure gel filtration chromatography (HPGFC) and matrix-assisted laser desorption ionization/time-of-flight mass (MALDI-TOF-MS) spectrometry method. The method of neutral red was carried out to investigate the phagocytosis of peritoneal macrophages. And Griess method and colorimetry were executed to detect the level of nitric oxide and iNOS activity respectively. Tumor necrosis factor-α and interleukin-6 were analyzed by enzyme linked immunosorbent assay (ELISA). Results Our results demonstrated that the subunit molecular weight of AGNP determined by SDS-PAGE was 15kD and the content of proteins determined by Bradford assay was 2.31mg/mL. The molecular weight of the AGNP was15, 114Da both of electrophoresis and MS purity. And the result of HPGFC showed that the molecular weight of AGNP was 31,086Da, Immunological studied indicated that AGNP could conspicuously increase phagocytosis of macrophages, facilitate the nitric oxide production, Tumor necrosis factor-α and interleukin-6 production. What is more, AGNP dose-dependently stimulated NO formation through the up-regulation of iNOS activity. Conclusions In conclusion, AGNP had good immunoregulatory effects supporting the traditional claims and may provide a valuable therapeutic strategy to promoting immune function and metabolism.

This protocol can be used to measure the binding capacity of chitin binding proteins and visualizing this using a Bradford assay. The protocol has been adapted from: F. Labroussaa, A. R. Zeilinger, and R. P. P. Almeida, “Blocking the Transmission of a Noncirculative Vector-Borne Plant Pathogenic Bacterium,” Mol. Plant-Microbe Interact., vol. 29, no. 7, pp. 535–544, Jul. 2016.

In polymer therapeutics, polymer-based carrier systems conjugated with antioxidants have been synthesized and studied to improve diagnosis and treatment of diseases, such as, in cancer and tumor. The natural bioactive compound hyaluronic acid (HA), which is essential in medical and pharmaceutical fields, is a linear polymer composed of repeating disaccharide units of β-1,3-N-acetyl glucosamine and β-1,4-glucuronic acid. In this study, HA fractions of enzyme-assisted glycosaminoglycans (GAGs) extract from Liparis tessellatus eggs were grafted with gallic acid (GA), caffeic acid (CA), and ferulic acid (FA) via a free radical-mediated method, and with nisin via amide bond formation. The modification has been confirmed through FTIR and 1H NMR spectroscopy and quantified by Folin-Ciocalteu and Bradford assay. FTIR spectra of grafted HA samples exhibited the typical phenolic characteristics within 1450–1650 cm−1, and the formation of amide bond in nisin-grafted HA was shown by absorption peak within 1545–1646 cm−1. 1H NMR spectra showed new peaks of phenyl protons at 6.3–7.7 ppm and new peaks at 0.9–2.9 ppm of amino acids residues protons. These results all confirmed the successful grafting of GA, CA, FA and nisin onto the HA backbone extracted from L. tessellatus eggs.

PurposeTo develop a magneto-nanosensor (MNS) based multiplex assay to measure protein and autoantibody biomarkers from human serum for prostate cancer (CaP) diagnosis.Materials and methodsA 4-panel MNS autoantibody assay and a MNS protein assay were developed and optimized in our labs. Using these assays, serum concentration of six biomarkers including prostate-specific antigen (PSA) protein, free/total PSA ratio, as well as four autoantibodies against Parkinson disease 7 (PARK7), TAR DNA-binding protein 43 (TARDBP), Talin 1 (TLN1), and Caldesmon 1 (CALD1) and were analyzed. Human serum samples from 99 patients (50 with non-cancer and 49 with clinically localized CaP) were evaluated.ResultsThe MNS assay showed excellent performance characteristics and no cross-reactivity. All autoantibody assays showed a statistically significant difference between CaP and non-cancer samples except for PARK7. The most significant difference was the combination of the four autoantibodies as a panel in addition to the free/total PSA ratio. This combination had the highest area under the curve (AUC)- 0.916 in ROC analysis.ConclusionsOur results suggest that this autoantibody panel along with PSA and free PSA have potential to segregate patients without cancer from those with prostate cancer with higher sensitivity and specificity than PSA alone.

Prions cause transmissible infectious diseases in humans and animals and have been found to be transmissible by blood transfusion even in the presymptomatic stage. However, the concentration of prions in body fluids such as blood and urine is extremely low, and therefore direct diagnostic tests on such specimens often yield false-negative results. Quantitative preanalytical prion enrichment may significantly improve the sensitivity of prion assays by concentrating trace amounts of prions from large volumes of body fluids. Here we show that beads conjugated to positively charged peptoids not only captured PrP aggregates from plasma of prion-infected hamsters, but also adsorbed prion infectivity in both the symptomatic and preclinical stages of the disease. Bead absorbed prion infectivity efficiently transmitted disease to transgenic indicator mice. We found that the readout of the peptoid-based misfolded protein assay (MPA) correlates closely with prion infectivity in vivo, thereby validating the MPA as a simple, quantitative, and sensitive surrogate indicator of the presence of prions. The reliable and sensitive detection of prions in plasma will enable a wide variety of applications in basic prion research and diagnostics.

Small molecule ligands, which can bind to a protein of interest with high affinity and specificity, are a burgeoning kind of recognition element in nano-inspired biosensors for protein assay. Similar to most other recognition molecules mentioned in previous Chapters, small molecule ligands are also unable to respond to target protein binding in a measurable way. To resolve this problem, rationally designed and engineered small molecule ligand-based probes, such as small molecule-tethered chemical probes and small molecule-linked DNA probes, have been rapidly developed and found successful applications in detecting disease protein biomarkers. This chapter aims to provide an overview of the latest advances in this field by detailing a number of representative biosensors according to the category of small molecule ligand-based probes.

Biotechnology progress (2019). doi:10.1002/btpr.2911, Published by Wiley, Malden, MA