Search

Gingival mesenchymal stem cell-conditioned medium (GMSC-CM) is a conditioned medium obtained from cultured gingival mesenchymal stem cells . GMSCs are easily isolated from gingival tissue, whereas gingival tissue is easily obtained by minimally invasive techniques. On the other hand, C M contains proteins, cytokines, chemokine s and growth factors that play an important role in osteogenic differentiation. This study aim s to determine the total protein and calcium levels in GMSC-CM. GMSCs were grown in culture media with 10% of FBS. CM of GM SC was obtained from the media collection process with a 0.22 µm filter and concentrated with a centrifugal filter to obtain a concentrated GMSC-CM. The concentration of total protein was performed by bicinchoninic acid protein assay on GMSC-CM and concentrated GMSC-CM. Calcium level was performed by atomic absorption spectroscopy method. The results are that GMSC-CM had a total protein concentration of 2502±0.06 µg/ml, and the concentrated GMSC-CM was 1.912±0.08 μg/ml. The calcium level of GMSC-CM was 0.0 09 % and concentrated GMSC-CM was 0.0 09 %. It can be concluded that GMSC-CM had a high concentration of total protein and calcium. These parameters were used to develop bioprocesses to enhance the production of GMSC-CM, which will support the implementation of cell-free therapy for tissue regeneration.

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.

Hepatitis B causes major deathly infections in developing countries. The surface antigen preS1, hepatocytes attachment site, plays a crucial role in development and progression of disease. The present study was, therefore, an effort to develop an efficient expression system for PreS1 gene in Esherishia coli. PreS1 gene was amplified and cloned in pTZ57R/T vector. Following confirmation through restriction enzyme digestion and sequencing, it was subcloned in pET22b(+). E. coli BL21 (DE3) CodonPlus cells were transformed with the recombinant plasmid. PreS1 gene was induced using IPTG, the protein was expressed and quantified with Bradford assay and analyzed on SDS-PAGE. Enzyme Linked Immunosorbent Assay and Western Blot analysis were performed for protein integrity and conformation.

Human epidermal growth factor receptor 2 (HER2) status in breast cancer is routinely determined through immunohistochemistry (IHC) and/or in situ hybridisation (ISH) performed on whole tissue sections (WS). The purpose was to evaluate whether a gene protein assay (GPA) combining IHC with ISH, performed on breast cancer tissue microarray (TMA), is suitable for large-scale retrospective HER2 status evaluation. TMAs from 606 tumours from a Swedish population-based cohort (2005–2012) were stained with GPA. GPA IHC on TMA yielded weaker staining than IHC on WS during routine pathological assessment (86.0% agreement). However, final HER2 status agreement between GPA on TMA and WS based on both IHC and ISH was 97.7%. Only 14 tumours were discordant and one tumour with IHC score 1+ on both TMA and WS was HER2 amplified on TMA. In conclusion, GPA on TMA is suitable for large-scale retrospective evaluation of HER2 status.

BACKGROUND As the only TB vaccine available, Bacillus Calmette-Guérin shows variable efficacy in adults and does not provide protection against the resuscitation of latent TB infections. Resuscitation-promoting factor B (RpfB) is a protein produced by Mycobacterium tuberculosis during the resuscitation phase and is promising as a novel TB vaccine. This study was aimed to analyze the immunogenicity of the gluthathione S-transferase (GST)-RpfB recombinant protein on mice splenocytes in vitro. METHODS After induction with isopropyl β-D-1-thiogalactopyranoside, the protein was extracted by sonication followed by solubilization in 8 M urea buffer. Protein was then re-natured and purified with a GST chromatography column. The isolated protein was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot using anti-GST antibodies, and its concentration was determined using the Bradford method. Each group of splenocytes was treated with 25 μg/ ml of the recombinant protein (GST-RpfB), GST, and phytohemagglutinin. Antigen induction was repeated twice at 24 and 72 hours. The supernatant was collected at 96 hours and interferon gamma (IFNγ), interleukin (IL-12, IL-4, and IL-10) levels were measured with enzyme-linked immunosorbent assays. RESULTS GST-RpfB recombinant proteins were expressed in the form of inclusion bodies with a molecular weight of approximately 66 kDa. Based on the independent t-test, GST-RpfB stimulated IFNγ and IL-12 production but not IL-4 and IL-10. CONCLUSIONS The GST-RpfB protein has been immunogenically proven and is a potential candidate as a novel subunit TB vaccine.

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.

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.

Hidayaturrahmah, Mabrur, Santoso HB, Sasmita R, Rahmy USA, Badruzsaufari. 2019. Short Communication: Protein profiles of Giant Mudskipper and its potential use as biomarker candidate for heavy metal contamination in Barito Estuary, Indonesia. Biodiversitas 20: 745-753. Giant mudskipper (Periphthalmodon schlosseri, Pallas 1770) is one of important biological diversity that potential as bio-indicator in environment assessment which is heavy metal contamination. These fish are susceptible to ambient environment, that effect to their physiological mechanism including protein synthesis and expression. This research aims to identify protein profiles of giant mudskipper muscle and review its potential application as a biomarker for heavy metal contamination in Barito Estuary of South Kalimantan. Total proteins were isolated from fish muscle using Tris EDTA buffer and then precipitated using Ammonium sulfate salt. The protein concentration was measured using Bradford assay and then separated based on molecular weight using the SDS-PAGE method. The result showed muscle proteins of giant mudskipper could be precipitated on optimum condition at Ammonium sulfate saturation 60-70% which protein concentration 5.106 mg.mL-1. The protein separated into 20 bands for raw and precipitated proteins which molecular weight ranging from 33 into 184 kDa. Further study is needed for protein identification based on amino acids sequence of protein bands for biomarker discovery and validation.

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.

Interferon gamma (IFN-γ) release assays (IGRAs) are increasingly used to test for latent tuberculosis (TB) infection. Although highly specific, IGRAs have a relatively high false-negative rate in active TB patients. A more sensitive assay is needed. IFN-γ-induced protein 10 (IP-10) is an alternative biomarker with a 100-fold-higher expression level than IFN-γ, allowing for different analysis platforms, including molecular detection. The PCR technique is already an integrated tool in most TB laboratories and, thus, an obvious platform to turn to. In this case-control study, we investigated the diagnostic sensitivity and specificity of a molecular assay detecting IP-10 mRNA expression following antigen stimulation of a blood sample. We included 89 TB patients and 99 healthy controls. Blood was drawn in QuantiFeron-TB gold in-tube (QFT) assay tubes. Eight hours poststimulation, IP-10 mRNA expression was analyzed, and 20 h poststimulation, IP-10 and IFN-γ protein plasma levels were analyzed using an in-house IP-10 enzyme-linked immunosorbent assay (ELISA) and the official QFT ELISA, respectively. The IP-10 mRNA assay provided high specificity (98%), sensitivity (80%), and area under the concentration-time curve (AUC) (0.97); however, the QFT assay provided a higher overall diagnostic potential, with specificity of 100%, sensitivity of 90%, and AUC of 0.99. The IP-10 protein assay performed on par with the QFT assay, with specificity of 98%, sensitivity of 87%, and AUC of 0.98. We have provided proof of high technical performance of a molecular assay detecting IP-10 mRNA expression. As a diagnostic tool, this assay would gain from further optimization, especially on the kinetics of IP-10 mRNA expression.

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.

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.

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.

The analysis of coccal lactic acid bacteria (CLAB) strains for their competence in polysaccharide production has been given proper attention as texturing and thickening agents. The characterization of these strains has been performed from two natural environments (camel milk and red meat). All the isolates were evaluated for exopolysaccharides (EPS) production on certain solid medium and ruthenium red milk agar plate. Based on their EPS-producing colony phenotype, five strains were chosen giving an important white-color and mucoid aspect on sucrose-based media which being the best for detecting the EPS. Quantitative estimation of EPS indicated that amount of this polymer rendered more than 400 mg/L and the apparent viscosity ranged from 2.1 to 2.9 milli Pascals per second (mPa.s). Therefore, there was not found a close relationship between the amount of EPS and the apparent viscosity. Three strains were selected for their significant production of EPS. For protein assay, a low content of protein was obtained on crude polymer revealing the quality of EPS extracts. Key words: Coccal lactic acid bacteria (CLAB), exopolysaccharides (EPS), sucrose-based medium, apparent viscosity, amount of EPS.

An effective double antibody sandwich ELISA (DAS-ELISA) method based on monoclonal (mAb) and chicken egg yolk IgY antibodies was developed to determine phosvitin (PV) content in therapeutic and functional products. Leghorn laying hens were immunized with purified PV to produce anti-PV IgY antibody in the egg yolk. High anti-PV IgY titer obtained from the egg yolks collected during 4–10 weeks of the immunization period contained approximately 6.2% of specific anti-PV IgY in total IgY. The PV detection range of the DAS-ELISA and biotinylated DAS-ELISA was 16.8–90 and 7.5–40 ng/mL, respectively. However, biotinylated DAS-ELISA was the better method for PV quantification in terms of accuracy and sensitivity. This highly efficient PV detection method may recuperate the performance of the existing protein assay methods as well as facilitate future research on PV bioactivities and applications.

About (20) Pseudomonas rogenosa isolate were experienced for their ability of protease production by calculating the diameter of lysis area after developing on skim milk agar medium (qualitatively ) andamp; the results exhibited that only isolate no (5), was higher isolate for protease making of (26mm) of lysis area. Then, the protein concentration also identified by Bradford method andamp; it was found of 0.16 mg/ ml , then purification was done by using an ion- exchange chromatography with DEAE sephadex G- 100 column andamp; the results showed the presence of 1 peak of enzyme with 50 Kd of molecular weight 2 peaks of other proteins . we tried to investigate the invitro Cytotoxic effect of purified enzyme against two human cancer lines, HeP2 (Human larynx epidermed carcinoma ) , RD ( Rabdo- Sarcoma ) , andamp; one normal cell line Ref ( Rat embryonic fibroblast ). The cancer andamp; normal cells were treated with different concentrations of protease enzyme ranging from ( 0.05, 0.1, 0.2, 0.4,0.8andamp; 0.16 mg/ml) then incubated for additional 48h at 37C 0 andamp; the results showed highest toxicity ( 80.28%) of protease enzyme on RD , moderate cytotoxicity (45.52%) on Hep andamp;slight toxicity ( 37.12% )on normal cell line (Ref) in a concentration (0.8mg/ml).

About (20) Pseudomonas rogenosa isolate were experienced for their ability of protease production by calculating the diameter of lysis area after developing on skim milk agar medium (qualitatively ) and the results exhibited that only isolate no (5), was higher isolate for protease making of (26mm) of lysis area. Then, the protein concentration also identified by Bradford method and it was found of 0.16 mg/ ml , then purification was done by using an ion- exchange chromatography with DEAE sephadex G- 100 column and the results showed the presence of 1 peak of enzyme with 50 Kd of molecular weight 2 peaks of other proteins . we tried to investigate the invitro Cytotoxic effect of purified enzyme against two human cancer lines, HeP2 (Human larynx epidermed carcinoma ) , RD ( Rabdo- Sarcoma ) , and one normal cell line Ref ( Rat embryonic fibroblast ) . The cancer and normal cells were treated with different concentrations of protease enzyme ranging from ( 0.05, 0.1, 0.2, 0.4,0.8and 0.16 mg/ml) then incubated for additional 48h at 37C0 and the results showed highest toxicity ( 80.28%) of protease enzyme on RD , moderate cytotoxicity (45.52%) on Hep andslight toxicity ( 37.12% ) on normal cell line (Ref) in a concentration (0.8mg/ml).

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.

Sasmita R, Mabrur, Rahmy USA, Badruzsaufari. 2019. Genetic diversity and phylogenetic relationship among Anabantoidei fish (Anabantiformes) in South Kalimantan, Indonesia based on SDS-PAGE analysis. Biodiversitas 20: 2519-2527. Protein profile is a molecular marker for diversity and phylogenetic analysis of germplasm, including Anabantoidei fish which is abundant in freshwater ecosystem of South Kalimantan. The aim of this research was to analyze genetic diversity and construct phylogenetic relationship among Anabantoidei fish based on SDS PAGE analysis. Protein was extracted from muscle tissue of six species of Anabantoidei fish and precipitated using Ammonium sulfate salt. Soluble protein content was determined using Bradford assay and then separated based on SDS-PAGE method. Genetic diversity and phylogenetic relationship were constructed using PAST software based on UPGMA method. The results showed that the soluble protein of fish muscle can be precipitated optimally in different ammonium sulfate concentration. Based on SDS-PAGE analysis, 59 different protein bands have been separated from gels with molecular weight ranging from 28.15 to 181.61 kDa. On the protein level, the Anabantoidei fish showed high genetic polymorphism (greater than 90%) with 3, 2 and 5 monomorphic bands on non-precipitated, AS-precipitated and combination between non- and AS-precipitated proteins, respectively. The phylogenetic reconstruction also exhibited that the Anabantoidei fish has the unique phylogenetic trees, especially for the combined protein datasets. This information would be useful for freshwater fish conservation and breeding programs.

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.

Background: Oxidative stress is an imbalance between reactive species and antioxidants production, leading to protein, DNA and lipid damage. The aim of this study was to evaluate the oxidative stress levels in buccal cells of ten non-shisha and heavy shisha smokers using MAWI iSWAB protein tubes. Materials and findings: The presence of S-glutathionylation, NOX2 and iNOS was analyzed in buccal cells of ten non-smokers and ten heavy shisha smokers using MAWI i-SWAB protein tubes, knowing that they were able to detect previously several protein oxidation biomarkers (S-nitrosylation and nitrotyrosine). Proteins concentrations were assessed by Bradford assay then western blot method was used to analyze the presence of the different proteins of interest. Conclusion: Results confirmed the findings about the ability of these tubes to detect S-nitrosylation, nitrotyrosine, as well as S-glutathionylation. Results showed also the presence of immature NOX2 in human buccal cells equally. Levels of iNOS were the same in non-shisha and heavy shisha smokerss.

Despite a clinical, economic, and regulatory imperative to develop companion diagnostics, precious few new biomarkers have been successfully translated into clinical use, due in part to inadequate protein assay technologies to support large-scale testing of hundreds of candidate biomarkers in formalin-fixed paraffin-embedded (FFPE) tissues. Although the feasibility of using targeted, multiple reaction monitoring mass spectrometry (MRM-MS) for quantitative analyses of FFPE tissues has been demonstrated, protocols have not been systematically optimized for robust quantification across a large number of analytes, nor has the performance of peptide immuno-MRM been evaluated. To address this gap, we used a test battery approach coupled to MRM-MS with the addition of stable isotope-labeled standard peptides (targeting 512 analytes) to quantitatively evaluate the performance of three extraction protocols in combination with three trypsin digestion protocols (i.e., nine processes). A process based on RapiGest buffer extraction and urea-based digestion was identified to enable similar quantitation results from FFPE and frozen tissues. Using the optimized protocols for MRM-based analysis of FFPE tissues, median precision was 11.4% (across 249 analytes). There was excellent correlation between measurements made on matched FFPE and frozen tissues, both for direct MRM analysis (R(2) = 0.94) and immuno-MRM (R(2) = 0.89). The optimized process enables highly reproducible, multiplex, standardizable, quantitative MRM in archival tissue specimens.

Enterohemorrhagic Escherichia coli are important human food-borne pathogens. Recently, Shiga toxin-producing E. coli (STEC) causes life-threatening hemolytic-uremic syndrome (HUS). In this study, Stx2B gene, a subunit of Shiga toxin, was amplified via polymerase chain reaction (PCR) from the chromosomal DNA of clinical fecal sample using appropriate primers. The PCR product was cloned to commercially available plasmid pH6HTN His6HaloTag® T7 containing two purification tags, namely, six histadine tag and Halo tag. The integrity of the constructed plasmid was confirmed using restriction enzyme mapping and sequencing. Then, Stx2B protein expressed after induction with isopropyl β-D-1-thiogalactopyranoside (IPTG) in E. coli JM109 (DE3) under the control of the T7 promotor. The two step purification trains were used to purify native Stx2B. First step purification was Ni-immobilized metal ion affinity chromatography (IMAC) column, followed by second step using HaloLink resin. The native Stx2B was obtained after column cleavage of halo-tag using HaloTEV protease. Maximum protein expression of Stx2B economically was obtained using 1 mM IPTG for 4 h at 37°C. Protein identity was confirmed by a band at ~11.4 kDa using 15% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and StxB2 yield was 450 µg ml-1 confirmed by Bradford assay. Recombinant Stx2B protein was produced in highly pure yield using HaloTag technology. Key words: Escherichia coli O157:H7, StxB gene, expression, HaloTag technology, purification.

Objective of the study is to deploy 2-D electrophoresis for the construction of lysate “protein profiles” of bacterial cultures of particularly dangerous infections. Materials and methods. “Protein profiles” are obtained on the model of non-toxigenic V. cholerae biovar El Tor M-888 strain and Y. pestis EV NIIEG strain, cultivated at 28 and 37 °C. Commercial sample of E. coli strain lysate serves as comparator (reference) product. SDS-PAGE-electrophoresis is carried out following classical Laemmli approach. 2D-electrophoresis is based on O’Farrel technique and manufacturer’s recommendations. For protein visualization, staining of electrophoregrams with coomassie blue G-250 and silver nitrate is used. Protein concentration (load) is evaluated with the help of Bradford protein assay. Cell lysate samples are purged of non-protein impurities applying Ready Prep 2-D Cleanup Kit. Results and conclusions. Described is a highly effective complex method of protein mixture separation, which assumes preparation of bacterial culture probes where cells are fractured under ultrasonic exposure in the lysing buffer, then purified and analyzed either in reference to the charge in pH gradient, or - to molecular mass in SDS-PAGE-electrophoresis. After the staining 2-D gels are assayed by means of “Dymension” software product, installed on the platform of multi-functional gel documentation system “Syngene”. Utilization of stripes with immobilized pH gradient of a preset range, commercial reagent kits, and large-sized polyacrylamide gels has allowed for optimization of the conditions for performing 2D-electrophoresis, reduction of the time elapsed, and improvement of accuracy and reproducibility. On the model of non-toxigenic V. cholerae biovar El Tor M-888 strain and Y. pestis EV NIIEG strain, cultivated at 28 and 37 °C, applying 2D-electrophoresis, obtained are the protein “profiles” of the investigated strains, demonstrated is high efficacy of the method and possibility of its deployment for studies of gene expression dynamics as regards agents of particularly dangerous infections.

Colorimetric protein assays, such as the Coomassie blue G-250 dye-binding (Bradford) and bicinchoninic acid (BCA) assays, are commonly used to quantify protein concentration. The accuracy of these assays depends on the amino acid composition. Because of the extensive use of reductive methylation in the study of proteins and the importance of biological methylation, it is necessary to evaluate the impact of lysyl methylation on the Bradford and BCA assays. Unmodified and reductively methylated proteins were analyzed using the absorbance at 280 nm to standardize the concentrations. Using model compounds, we demonstrate that the dimethylation of lysyl ε-amines does not affect the proteins' molar extinction coefficients at 280 nm. For the Bradford assay, the responses (absorbance per unit concentration) of the unmodified and reductively methylated proteins were similar, with a slight decrease in the response upon methylation. For the BCA assay, the responses of the reductively methylated proteins were consistently higher, overestimating the concentrations of the methylated proteins. The enhanced color formation in the BCA assay may be due to the lower acid dissociation constants of the lysyl ε-dimethylamines compared with the unmodified ε-amine, favoring Cu(II) binding in biuret-like complexes. The implications for the analysis of biologically methylated samples are discussed.

Protein quantification is essential in a great variety of biochemical assays, yet the inherent systematic errors associated with the concentration determination of intrinsically disordered proteins (IDPs) using classical methods are hardly appreciated. Routinely used assays for protein quantification, such as the Bradford assay or ultraviolet absorbance at 280 nm, usually seriously misestimate the concentrations of IDPs due to their distinct and variable amino acid composition. Therefore, dependable method(s) have to be worked out/adopted for this task. By comparison to elemental analysis as the gold standard, we show through the example of four globular proteins and nine IDPs that the ninhydrin assay and the commercial QubitTM Protein Assay provide reliable data on IDP quantity. However, as IDPs can show extreme variation in amino acid composition and physical features not necessarily covered by our examples, even these techniques should only be used for IDPs following standardization. The far-reaching implications of these simple observations are demonstrated through two examples: (i) circular dichroism spectrum deconvolution, and (ii) receptor-ligand affinity determination. These actual comparative examples illustrate the potential errors that can be incorporated into the biophysical parameters of IDPs, due to systematic misestimation of their concentration. This leads to inaccurate description of IDP functions.

Introducción: El presente trabajo tuvo como objetivo identificar las proteínas implicadas en la solubilizacion de fosfatos inorgánicos (Pi) en Klebsiella aerogenes y evaluar la capacidad de solubilizacion de Pi bajo la influencia de fuentes de carbono (C) y nitrógeno (N) en bacterias aisladas de la Comarca Lagunera.Método: Se trabajó con ocho cepas bacterianas aisladas de cultivos forrajeros de La Comarca Lagunera y conservadas en glicerol a -20 °C. Se determinó el Índice de Solubilización (IS) en medio sólido Pikovskaya (PVK) y se cuantificó la solubilización de Pi en medio líquido con diferentes fuentes de C, N y fósforo (P). Se realizó una identificación molecular de la cepa que presentó la mayor capacidad para solubilizar el Pi. Para los estudios de expresión de proteínas, se realizaron cultivos bacterianos por duplicado con dos diferentes fuentes de Pi {(fosfato de calcio (FC) y fosfato de aluminio (FA)} en medio líquido PVK. Se llevó a cabo una precipitación de proteínas con ácido tricloroacético (TCA) y una cuantificación con BCATM Protein assay kit. Las proteínas se corrieron en gel de poliacrilamida al 12%, se realizó una digestión con la enzima tripsina y se analizaron los péptidos obtenidos por HPLC-espectrometría de masas. La identificación de la función de las proteínas se llevó a cabo mediante el uso de herramientas bioinformáticas.Resultados: K. aerogenes fue la bacteria que presentó una mayor capacidad para solubilizar FC (2.31 y 2.59) en medio sólido PVK Glucosa (G)-Urea (U) y G- Nitrato de Amonio (NA) respectivamente, FC (1.97 ppm) y FA (1.36 ppm) en medio líquido PVK U-G. En los cultivos bacterianos se identificaron doscientas dieciséis proteínas entre las cuales se encontraron: ABC transportadoras, histidina quinasa Pho, proteína del sistema de transporte específico de fosfato Pho U, involucradas con el aprovechamiento del P.Discusión o Conclusión: Las fuentes de C y N influyen en la capacidad de solubilización de Pi en bacterias nativas de La Comarca Lagunera. Los mejores resultados de solubilización se presentaron cuando la G se encontraba en el medio como fuente de C y la U como fuente de N. Bajo las condiciones de experimentación, no se observaron proteínas únicas de K. aerogenes para solubilizar el FC y FA. Se observó únicamente diferencias en el nivel de expresión de algunas proteínas identificadas.

Several, novel and different spectrophotometric methods based on UV and first-order derivative spectrophotometry, were developed to increase accuracy and improve sensitivity of the Bradford assay for protein quantification in hair samples. The linear calibration function was established in concentration range 2–90 μg/mL at pH 0.6. Significant improvement over the classical method was observed using three different methods based on first-order derivative spectra: signals at 550 nm (1D550), numerically integrated area under curve from 545 to 565 nm (AUC1D545-565) and the ratio of peaks at 550 and 420 nm (1D550/1D420). The correlation coefficients were 0.9924, 0.9932 and 0.9991, respectively. Accordingly, the best correlation was observed by the 1D550/1D420 ratio method. The LOD of 15.67 μg/mL (1D550), 14.80 μg/mL (AUC1D545-565), 5.45 μg/mL (1D550/1D420) and LOQ of 47.48 μg/mL (1D550), 44.85 μg/mL (AUC1D545-565), 16.51 μg/mL (1D550/1D420), were calculated. The sample recoveries for 1D550/1D420 ratio method have proven that its accuracy (95.05%) is within the acceptable interval of recoveries. The obtained 1.55% RSD for precision fell well within the criteria accepted in bioanalytical method validation. This method was applied for determination of total proteins in hair samples. Also, the influence of different chemical treatments on total protein amount in hair sample was investigated.

International audience; Sodium dodecyl sulfate electrophoresis (SDS) is a protein separation technique widely used, for example, prior to immunoblotting. Samples are usually prepared in a buffer containing both high concentrations of reducers and high concentrations of SDS. This conjunction renders the samples incompatible with common protein assays. By chelating the SDS, cyclodextrins make the use of simple, dye-based colorimetric assays possible. In this paper, we describe the optimization of the assay, focussing on the cyclodextrin/SDS ratio and the use of commercial assay reagents. The adaptation of the assay to a microplate format and using other detergent-containing conventional extraction buffers is also described.

Oxidative stress is the result of an imbalance in pro-oxidant/antioxidant homeostasis in favor of oxidants leading to the oxidation of DNA, lipids, and proteins. In this experiment, we investigated the oxidative profile of iSWAB-Mawi protein tubes by examining their ability to detect the protein oxidation biomarkers: nitrotyrosine and S-nitrosocysteine in buccal cells in a sample of 40 participants, equally divided between Lebanese University and Benta Pharma Industries. Protein concentrations were determined by Bradford assay in order to then be analyzed for the presence of nitrotyrosine and S-nitrosocysteine by western blot. Results showed the presence of S-nitrosocysteine and nitrotyrosine in buccal cells reflecting the ability of iSWAB-Mawi protein tubes to detect those protein oxidation biomarkers.

The major capsid protein (MCP) is the main immunogenic protein of iridoviruses, that has been widely used as an immunogen in vaccination trials. In this study, the codon-optimized giant salamander iridovirus (GSIV) MCP gene (O-MCP) was synthesized and cloned into a pPICZα B vector for secretory expression in the methylotrophic yeast Pichia pastoris after methanol induction. The expression of the O-MCP protein was detected by the Bradford protein assay, SDS-PAGE, Western blotting and electron microscopy. The Bradford protein assay indicated that the concentration of the O-MCP expressed was about 40 μg/ml in culture supernatants. SDS-PAGE analysis revealed that the O-MCP had a molecular weight of about 66 kDa and reacted with a His-specific MAb that was confirmed by Western blotting. Electron microscopy observations revealed that the purified O-MCP could self-assemble into virus-like particles. Healthy giant salamanders were vaccinated by intramuscular injection with the O-MCP antigen at a dose of 20 μg/individual. The numbers of erythrocytes and leukocytes in the peripheral blood of immunized Chinese giant salamanders increased significantly at day 3 and reached a peak at day 5 post-immunization. Meanwhile, the differential leukocyte counts of monocytes and neutrophils increased significantly at day 5 post-immunization compared to that of the control group. The percentage of lymphocytes was 71.33 ± 3.57% at day 21 post-immunization. The neutralization assay showed that the serum neutralizing antibody titer reached 321 at day 21 post-immunization. The GSIV challenge test revealed that the relative percent survival of Chinese giant salamanders vaccinated with O-MCP was 78%. These results indicated that the O-MCP antigen expressed by the Pichia pastoris system elicited significant immune response in the Chinese giant salamander against GSIV and might represent a potential yeast-derived vaccine candidate that could be used for the control of disease caused by the giant salamander iridovirus.

Quantitative protein analysis is routinely performed in clinical chemistry laboratories for diagnosis, therapeutic monitoring, and prognosis. Today, protein assays are mostly performed either with non-specific detection methods or immunoassays. Mass spectrometry (MS) is a very specific analytical method potentially very well suited for clinical laboratories. Its unique advantage relies in the high specificity of the detection. Any protein sequence variant, the presence of a post-translational modification or degradation will differ in mass and structure, and these differences will appear in the mass spectrum of the protein. On the other hand, protein MS is a relatively young technique, demanding specialized personnel and expensive instrumentation. Many scientists and opinion leaders predict MS to replace immunoassays for routine protein analysis, but there are only few protein MS applications routinely used in clinical chemistry laboratories today. The present review consists of a didactical introduction summarizing the pros and cons of MS assays compared to immunoassays, the different instrumentations, and various MS protein assays that have been proposed and/or are used in clinical laboratories. An important distinction is made between full length protein analysis (top-down method) and peptide analysis after enzymatic digestion of the proteins (bottom-up method) and its implication for the protein assay. The document ends with an outlook on what type of analyses could be used in the future, and for what type of applications MS has a clear advantage compared to immunoassays.

1H-1,2,3-Triazolylbenzohydrazides (6a-h and 11a-l) were synthesized from 2-chloronicotinaldehydes and evaluated for anti-mycobacterial activity against Mycobacterium tuberculosis H37Rv strain (ATCC-27294). Seven compounds 6b, 6e,f, 11d, 11h, 11j and 11l displayed potent anti-mycobacterial activity (MIC 2.8-6.2 μM). Potent anti-mycobacterial compounds were chosen for cytotoxicity studies by MTT protein assay against normal cell lines (PBMC and Raw 264.7) and shown low cytotoxicity. This is the first Letter assigning anti-mycobacterial activity, cytotoxicity and structure activity relationship for 1H-1,2,3-triazolylbenzohydrazides.

Objectives: The combined gene protein assay (GPA) can simultaneously assess HER2 gene copy number and protein on a single slide using bright-field microscopy. Methods: GPA was compared with a fluorescence in situ hybridization (FISH) assay on 50 invasive breast carcinomas with a 2+ score on immunohistochemistry (IHC). Results: The cases were categorized into positive, equivocal, or negative for HER2 gene amplification using the 2013 American Society of Clinical Oncology/College of American Pathologists criteria. This resulted in 82% agreement (41 of 50) between FISH and GPA. In addition, 25 known IHC 3+ breast carcinomas analyzed by GPA showed protein overexpression and clusters of HER2 gene consistent with unequivocal amplification, and 22 known IHC-negative cases were negative for HER2 gene amplification by GPA. Conclusions: Although GPA is an alternative to both IHC and FISH, it may be an unnecessary test for IHC 0/1+/3+ cases. The clinical utility of GPA appears to be similar to other in situ hybridization assays (ie, adjudicator of HER2 status for IHC 2+ cases).

A new solid-phase protein nano-assay is suggested for simple and sensitive estimation of protein content in sample buffers (a 1-μl sample is sufficient for analysis). The assay is different from conventional “on-filter” assays in that it uses inexpensive fully transparent polyacrylamide gel (PAAG)-coated glass plates as solid support and, thus, combines the convenience of “on-membrane” staining with the sensitivity and ease of documentation of “in-gel” staining (and, therefore, is especially suited for standard lab gel documentation systems). The PAAG plates assay is compatible with all dyes for in-gel protein staining. Depending on the sensitivity of the staining protocol, the assay can be used in macro-, micro-, and nano-assay formats. We also describe a low-cost two-component colloidal Coomassie brilliant blue G-250 (CBB G-250) staining protocol for fast quantitative visualization of proteins spotted on a PAAG plate (the detection limit is up to 2 ng of proteins even when using a Nikon CoolPix digital camera and white light transilluminator instead of a gel scanner). The suggested colloidal CBB G-250 protocol could also be used for visualizing nano-amounts of proteins in polyacrylamide gels. The PAAG plate assay could be useful for proteomic applications and, in general, for all cases where a fast, sensitive, and easily documentable cost-effective solid-phase protein assay is required.

Silk fibroin (SF) is a protein with bulky hydrophobic domains and can be easily purified as sericin-free silk-based biomaterial. Silk fibroin modified chitosan nanoparticle (SF-CSNP), a biocompatible material, has been widely used as a potential drug delivery system. Our current investigation studied the bio-effects of the SF-CSNP uptake by liver cells. In this experiment, the characterizations of SF-CSNPs were measured by particle size analysis and protein assay. The average size of the SF-CSNP was 311.9 ± 10.7 nm, and the average zeta potential was +13.33 ± 0.3 mV. The SF coating on the SF-CSNP was 6.27 ± 0.17 μg/mL. Moreover, using proteomic approaches, several proteins involved in the ubiquitin proteasome pathway were identified by analysis of differential protein expressions of HepG2 cell uptake the SF-CSNP. Our experimental results have demonstrated that the SF-CSNP may be involved in liver cancer cell survival and proliferation.

The aims of this work were to: i) purify GST-fusion protein from bacterial cell extracts of Escherichia coli; ii) quantify the protein by SDS PAGE and Bradford assay; iii) determine protein-DNA interaction of the purified protein by Electrophoretic Mobility Shift Assay. Bacterial culture prepared by inoculation of a single E. coli colony that had a GST fusion protein (gst: six-X10 hd) constructed by ligation of the six-7-hd (X10) sequence into the BamHI and EcoRI sites of the vector pGEX-2T, grown overnight, was sonicated using Cole-Palmer Ultrasonic Homogenizer. Fusion protein was eluted from the beads with Tris-glutathione buffer (50 mM Tris [pH 8.1], 20 mM glutathione), which contained reduced Glutathione. SDS-PAGE was used to calculate the extracted bound protein. Total protein quantification was then estimated by the Bradford assay. Bovine Serum Albumin (BSA) absorbance values were used to plot the standard curve used to calculate the concentrations of the sample proteins. Nylon membrane was used for the electrophoretic transfer; membrane was cross linked and detected by Pierce’s Chemiluminescent Nucleic Acid Detection module. Results showed that X10 gave a strong band shift observed in Lanes 6 and 7 for both 200 ng and 400 ng elute 1 samples; however, there was no shift in the bands for the wild-type, positive control. The concentration of the elute 1 was obtained by the Bradford assay as 242.52 ng/μl and that of elute 2 was 106.30 ng/μl. Similarly, the result obtained by gel analysis was 300 ng/μl (0.3 μg/μl) and 150 ng/μl (0.15 μg/μl) for elutes 1 and 2 respectively.

The aim of this study was to achieve high-level production of the human growth hormone (hGH) in the prokaryotic expression system. In this regard, we performed cloning, expression, and purification of a synthetic hGH gene in BL21 (DE3) strain of E. coli. The hGH production was determined by SDS-PAGE and western blotting techniques, and then the protein concentration was determined by the Bradford assay. To gain insight into the effect of different nutrients on the growth of E. coli and hGH production, in a preliminary assessment nine different types of the basal medium were analyzed. The highest growth of E. coli and hGH production were observed in TB and SOB media. Accordingly, design of experiments was employed for screening the most significant nutrients, and central composite face design was applied for the optimization. The optimum medium consisted of yeast extract (10 g/L), tryptone (10 g/L), and K2HPO4 (2 g/L). The optimum hGH concentration was 391 mg/L, which was 3-fold higher than the hGH concentration in the LB basal medium (119 mg/L). This production rate is the highest hGH concentration reported in the IPTG-inducible expression systems.

Background A number of factors regarding blood collection, handling and storage may affect sample quality. The purpose of this study was to assess the impact on plasma protein profiles by delayed centrifugation and plasma separation and multiple freeze-thaw cycles. Methods Blood samples drawn from 16 healthy individuals were collected into ethylenediaminetetraacetic acid tubes and kept either at 4 °C or 22 °C for 1–36 h prior to centrifugation. Plasma samples prepared 1 h after venipuncture were also subjected to two to eight cycles of freezing at −80 °C and thawing at 22 °C. Multiplex proximity extension assay, an antibody-based protein assay, was used to investigate the influence on plasma proteins. Results Up to 36 h delay before blood centrifugation resulted in significant increases of 16 and 40 out of 139 detectable proteins in samples kept at 4 °C or 22 °C, respectively. Some increases became noticeable after 8 h delay at 4 °C but already after 1 h at 22 °C. For samples stored at 4 °C, epidermal growth factor (EGF), NF-kappa-B essential modulator, SRC, interleukin 16 and CD6 increased the most, whereas the five most significantly increased proteins after storage at 22 °C were CD40 antigen ligand (CD40-L), EGF, platelet-derived growth factor subunit B, C-X-C motif chemokine ligand 5 and matrix metallopeptidase 1 (MMP1). Only matrix metallopeptidase 7 (MMP7) decreased significantly over time and only after storage at 22 °C. No protein levels were found to be significantly affected by up to eight freeze-thaw cycles. Conclusions Plasma should be prepared from blood after a limited precentrifugation delay at a refrigerated temperature. By contrast, the influence by several freeze-thaw cycles on detectable protein levels in plasma was negligible.

Haemonchus (H.) contortus is a blood feeding gastrointestinal nematode, and is considered one of the major threats for goat health and production globally. Crude somatic antigens (CSA) have been reported as sources of immunogens and provide better level of immunity against gastrointestinal nematodes. The present study aimed to quantify CSA of H. contortus worms (n=25) of both sexes followed by isolation of peptides through Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and their characterization. The triturate contained proteins having concentration of 1.3mg/ml as measured through Bradford assay. Seven different bands were appeared on gel ranging from 35KDa to 170KDa. The future prospects may include identification of the immunogenic peptides which can be used as vaccine candidates against H. contortus.

This is the protocolto be used in the determination of total protein in braintissue by the Pierce BCA method. BSA (Bovine Serum Albumin) is used as the standard and samples are prepared by sonification in 1% (w/v) SDS.

OBJECTIVE To assess the role of Rho/Rho-kinase pathway in the pathogenesis of cholesteatoma. MATERIALS AND METHODS Thirty-eight patients with cholesteatoma, who had gone mastoidectomies were enrolled in this prospective study. Cholesteatomas matrix (CM) and a piece of the external ear canal skin (EECS as control) were taken and transferred to the liquid nitrogen and kept at -86 °C for Rho A and Rho-kinase (ROCK) analysis with Western blotting and commercial ELISA kits (Cell Biolabs Inc., San Diego, CA). The tissues were homogenized by an appropriate ice-cold lysis buffer. Following centrifugation, the supernatant was taken and total protein amount was detected by the Bradford method. Thereafter, tissue homogenates were subjected to sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis electrophoresis then transferred to nitrocellulose membrane where it was treated with specific monoclonal primary antibody against to ROCK-2 and HRP-conjugated seconder antibody, respectively. The protein blots were visualized with commercial x-ray film and dansitometrically analyzed by the Scion Image Program (Cell Biolabs Inc., San Diego, CA). In another series of experiments, Rho-kinase activities were assessed by ROCK-2 ELISA kits. RESULTS There were no statistical differences in Rho A translocation between CM and EECS. However, ROCK activity was found to be lower in CM than EECS as detected by ELISA kits. Furthermore, ROCK protein expression was also significantly lower in CM than EECS as demonstrated by Western blotting. CONCLUSION Given Rho-kinase could take essential roles in cell differentiation, the results of this study implicate that down-regulated Rho-kinase could be responsible for the keratinocyte undifferentiation seen in cholesteatoma pathogenesis.

4-Dimethylaminoazobenzene (DAB, methyl yellow, or butter yellow), a human carcinogen, has been banned for use in foods since 1988. In 2014, DAB adulteration in Tofu occurred in Taiwan. We hypothesize that DAB can form [DAB•SBP]adduct adduct with soybean protein (SBP) which could damage Gastro-Duodenal-Hepatic axis. Sprague-Dawley rats gavage fed [DAB•SBP]adduct adduct revealed severely reduced body weight and damaged duodenum, liver, hepatic mitochondria, and spleen. Hepatic levels of glutathione and ATP were severely reduced. Serum GOT and GPT were substantially elevated. Analysis by the adsorption isotherm clearly revealed DAB formed very stable [DAB•SBP]adduct adduct at 1:1 molar ration (Phase A). The equilibrium constant of this colloidal adduct [DAB•SBP]adduct was KeqA = ∝, behaving as the most stable and toxic species. At higher protein concentration (Phase C) it formed conjugate [DAB×SBPgross]conjugate, with KeqC = 3.23×10−2 mg/mL, implicating a moderately strong adsorption. The in vitro pepsin digestibility test showed apparently reduced digestibility by 27% (by Ninhydrin assay) or 8% (by Bradford assay). Conclusively, this is the first report indicating that [DAB•SBP]adduct potentially is capable to damage the Gastro-Duodenal-Hepatic axis.

A basic Bronchoalveolar lavage (BAL) procedure in mouse is described here. Cells and fluids obtained from BAL can be analyzed by Hema3-staining, immunostaining, Fluorescence-activated cell sorting (FACS), PCR, bicinchoninic acid protein assay, enzyme-linked immunosorbent assay (ELISA), luminex assays, etc., to examine the immune cells, pathogens, proteins such as cytokines/chemokines, and the expression levels of inflammation-related and other genes in the cells. This will help to understand the underlying mechanisms of these lung diseases and develop specific and effective drugs.

Genetically induced outer membrane particles from Gram-negative bacteria, called Generalized Modules for Membrane Antigens (GMMA), are being investigated as vaccines. Rapid methods are required for estimating the protein content for in-process assays during production. Since GMMA are complex biological structures containing lipid and polysaccharide as well as protein, protein determinations are not necessarily straightforward. We compared protein quantification by Bradford, Lowry, and Non-Interfering assays using bovine serum albumin (BSA) as standard with quantitative amino acid (AA) analysis, the most accurate currently available method for protein quantification. The Lowry assay has the lowest inter- and intra-assay variation and gives the best linearity between protein amount and absorbance. In all three assays, the color yield (optical density per mass of protein) of GMMA was markedly different from that of BSA with a ratio of approximately 4 for the Bradford assay, and highly variable between different GMMA; and approximately 0.7 for the Lowry and Non-Interfering assays, highlighting the need for calibrating the standard used in the colorimetric assay against GMMA quantified by AA analysis. In terms of a combination of ease, reproducibility, and proportionality of protein measurement, and comparability between samples, the Lowry assay was superior to Bradford and Non-Interfering assays for GMMA quantification.

Secretomic analysis requires removal of serum proteins from cell-culture media. We evaluate the proteins washed from cells prepared in bovine serum-supplemented medium. PBS and serum-free-medium (SFM) were the washing solutions. A Bradford assay was used for total protein concentration and a 1D gel and LC-MS/MS, to assign the protein to human or bovine origin. For both wash solutions, all bovine protein had been removed by the third wash, without compromising the number of living cells. Further washes reduced the number of living cells, especially when using PBS. Proteomic analysis of wash supernatant showed that SFM induced greater lysis of dead cells. Three washes were sufficient to minimize the effects on cell viability, while still removing serum proteins. Washing in SFM resulted in contamination of the wash supernatant with lysed dead cell proteins. Washed cells were incubated in SFM and exposed to ionizing radiation. Analysis of the supernatant showed an increase in human cytoplasmic, plasma membrane, and nuclear protein following irradiation. Secreted proteins were also detected, but in smaller quantities. The significance of these findings extend to in vitro studies of bystander phenomena, since the proteins of lysed dead cells may participate in driving bystander responses.

Cisplatin-encapsulating maleimide-polyethylene glycol- Poly(d,l-lactic-co-glycolide) (cis-encapsulating mal-PEG-PLGA) particles were produced using the electrospray technique and bioconjugated with CD44 monoclonal antibody, targeting the counterpart receptor. The produced suspension of cis-encapsulating CD44-PEG-PLGA particles contains an antibody loading of 12.65-15.17 μg/mL and efficiently targets a CD44-overexpressed ovarian cancer cell line, such as CP70 and SKOV-3, within 6h of treatment, which was determined by Bradford assay, immunofluorescence analysis, and confocal laser scanning microscopic (CLSM) study. Most importantly, no tedious multi-step bioconjugation procedures are needed to synthesize mal-PEG-PLGA vehicles for antibody and drug loading, avoiding the undesirable hydrolysis of mal-PEG moiety and so successfully generating the cis-encapsulating mal-PEG-PLGA particles within one step. After conjugation of the CD44 antibody, the produced cis-encapsulating CD44-PEG-PLGA particles exhibited a better anti-proliferative ability against ovarian cancer cells compared to free form of cisplatin and PLGA particles without CD44 conjugation. Notably, the cis-encapsulating CD44-PEG-PLGA particles have approximately 10-14% greater the anti-proliferative ability against CP70 and SKOV-3 cells at a concentration of 1.25 μM, which falls within the concentrations used in chemotherapy. The proposed antibody-functionalization strategy represents an excellent platform for preparing particles with targeting ability in cancer therapy in vitro or in vivo.

In this paper we present a modified and improved protein assay that was previously described as ‘amidoschwarz assay’ by Schaffner and Weissmann (Anal. Biochem. 56, 1973, 502–514). Our improved protein assay is user-friendly and 30 to 40 times more sensitive than the earlier method. The assay was developed into 3 formats (maco, micro, and nanoassay) with TCA as protein precipitating agent; measuring up to 96 samples. The macro and micro formats of this assay require a single reagent staining with amido black of protein dots, bound to nitrocellulose membrane with lowest protein measurements to 1 μg and 0.1 μg respectively. The nanoassay on the other hand with combination staining of amido black followed by colloidal gold can extend the detection limit to 2.5 ng of protein. Protein concentrations were determined by densitometry and/or spectrophotometry. This assay is compatible with many ionic and non-ionic detergents. This improved protein assay provides an additional choice to researchers in measuring total protein concentration accurately in dilute biological samples as low as 0.125 μg/ml, prior to their biochemical analysis such as in comparative proteomics.

Helicobacter pylori colonizes in the stomach of nearly half of the world's population. It is one of the most common causes of chronic bacterial infection and a well-known risk factor for gastric cancer in humans. This study aimed to evaluate the cloning, expression, and purification of the HpaA-CagA fusion recombinant protein of H. pylori in E. coli BL21 (DE3) strain. The HpaA-CagA construct was designed using bioinformatics tools. The construct was then sub-cloned to the pET21b prokaryotic expression vector. Then, E. coli BL21 (DE3) was transfected with the pET21b-HpaA-CagA. Then, E. coli BL21 (DE3) was transformed with pET21b-HpaA-CagA. The gene expression was induced by IPTG. The protein expression was verified using SDS page and western blot analysis. The recombinant protein was finally purified using nickel nitrilotriacetic acid (Ni-NTA) resin. The waste materials were removed by dialysis. The concentration of recombinant protein was determined by the Bicinchoninic Acid Protein Assay Kit (Parstoos). In this study, HpaA-CagA was sub-cloned into the pET21b vector. The expression of the recombinant protein was confirmed in the host. The recombinant protein with the weight of 69 kDa was successfully purified. We here presented an efficient method to construct, clone, express, and purify the HpaA-CagA recombinant protein. The recombinant HpaA-CagA protein showed immunogenicity feature with human antiserum.