PAGE, polyacrylamide gel electrophoresis Dotaz Zobrazit nápovědu
The nonradioactive method, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) in the presence of Phos-tag (Phos-tag electrophoresis), is used to evaluate a kinase autophosphorylation and/or phosphotransfer reaction from a kinase/ATP to its protein substrate. This method outperforms radioisotope methods using [32P]ATP for detecting trace amounts of phosphorylated protein in fresh protein preparations. Phos-tag electrophoresis has been used to perform detailed analyses of the kinase activity of a heme-based oxygen sensor-specifically, a globin-coupled histidine kinase from the soil bacterium Anaeromyxobacter sp. Fw109-5 (AfGcHK).
Introduction: Thalassemia is a healthcare challenging disease all over the world. It imparts a great burden on patients' families and healthcare institutions. Scientists focus on new aspects to overcome these challenges and increase patient tolerance of disease complications. This study aims to quantify β-Hydroxybutyrate Dehydrogenase BHBDH activity in thalassemia patients compared to the control group and their correlation with the patient's demographic characteristics.Methods: To do so, serum was collected from patients and the control group and analyzed biochemically for targeted laboratory tests. We determined β-Hydroxybutyrate Dehydrogenase from normal human serum using biochemical molecular techniques.Results: The results showed that BHBDH activity is significantly higher in the patients compared to the control group regardless of age, sex, or marital status. The results confirmed that enzyme activity and the purification folds were (0.0214 U/ml) and (51.7) respectively for the partially purified enzyme. Furthermore, the proportional molecular weight of the incompletely isolated β-Hydroxybutyrate Dehydrogenase was (125.8±0.5 kDa) using gel filtration chromatography. The comparative molecular weight of the subunit of partially isolated β-Hydroxybutyrate Dehydrogenase was (32.1±0.5 kDa) using SDS-PAGE.Conclusion: we demonstrate that BHBDH enzymatic activity is higher than control and this could be a prognostic or diagnostic tool in thalassemia patients.
- MeSH
- beta-talasemie * krev MeSH
- biochemická analýza krve metody MeSH
- diagnostické techniky molekulární metody MeSH
- elektroforéza v polyakrylamidovém gelu MeSH
- gelová chromatografie MeSH
- hydroxybutyrátdehydrogenasa * chemie fyziologie izolace a purifikace krev MeSH
- lidé MeSH
- molekulová hmotnost MeSH
- statistika jako téma MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- klinická studie MeSH
- Geografické názvy
- Irák MeSH
Cryptosporidium parvum infects enterocytes in diverse vertebrates, including humans, and causes diarrheal illness. However, no effective drugs are available for this protozoan infection. The P23 protein of C. parvum is a protective antigen, considered a potential candidate for developing an effective vaccine against cryptosporidiosis. In this study, the complementary DNA (cDNA) of the p23 gene was subcloned to Escherichia coli DH5α, with one nucleotide difference. The constructed plasmid pNZ8149-P23 was transferred by electroporation to Lactococcus lactis NZ3900, and the recombinant L. lactis NZ3900/pNZ8149-P23 strain was screened in Elliker-medium by adding bromocresolpurple indicator. A 23-kDa protein was detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) after nisin induction in LM17 broth medium, suggesting that P23 protein was in the form of glycosylation. Simultaneously, an optimal induction time of 9 h was determined, and the density of OD600 = 2.7 was tested. Through western blot and indirect immunofluorescence (IIF) analysis, the immunocompetence of expressed P23 antigen was identified, and its location of release to the cell interior of recombinant L. lactis was manifested. The first report of a food-grade genetically engineered L. lactis strain expressing a P23 antigen of C. parvum is herein presented. This result provides a novel and safe utilization method of P23 against C. parvum infection.
- MeSH
- Cryptosporidium parvum * genetika metabolismus MeSH
- Cryptosporidium * metabolismus MeSH
- kryptosporidióza * prevence a kontrola MeSH
- Lactococcus lactis * genetika metabolismus MeSH
- lidé MeSH
- pyridinolkarbamát MeSH
- rekombinantní proteiny genetika metabolismus MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
New strategies for the fast, efficient, and environmentally friendly extraction of proteins are required to isolate desired bioactive compounds from a technological point of view. In this study, utilization of the pressurized water extraction (PWE) at low temperature (40 °C) for isolation of mistletoe proteins was investigated. PWE effectiveness, based on protein fingerprints, were compared with those obtained by conventional extractions using 10 mmol L-1 Tris-HCl buffer pH 8.3, 50 mmol L-1 phosphate buffer pH 7, or deionized water. The extracts were precipitated using acetone, trichloroacetic acid (TCA), and 20% (w/v) TCA/acetone and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. PWE was more or equally efficient for isolation of mistletoe proteins than evaluated conventional extraction methods. The proteomic analysis combining mass spectrometry and database searching confirmed the presence of 35 proteins in PWE extracts precipitated by acetone, which was the most compounds identified from all studied extracts. The PWE high extraction power was revealed for multiple viscotoxin isoforms and specific enzymes indispensable for the synthesis of terpenes.
- MeSH
- elektroforéza v polyakrylamidovém gelu MeSH
- listy rostlin MeSH
- proteomika MeSH
- Viscum album * MeSH
- voda MeSH
- Publikační typ
- časopisecké články MeSH
Nitrotyrosine formation is caused by presence of reactive oxygen and nitrogen species. Nitration is a very selective process leading to specific modification of only a few tyrosines in protein molecule. 2D electrophoresis and western blotting techniques coupled with mass spectrometry are common methods used in analysis of proteome. Here we describe protocol for analysis of peroxynitrite-induced protein nitration in isolated mitochondria. Mitochondrial proteins are separated by 2D electrophoresis and transferred to nitrocellulose membrane. Membranes are then incubated with antibodies against nitrotyrosine. Positive spots are compared with corresponding Coomassie-stained gels, and protein nitration is confirmed with mass spectrometry techniques.
- MeSH
- 2D gelová elektroforéza metody MeSH
- hmotnostní spektrometrie metody MeSH
- imunoblotting metody MeSH
- kyselina peroxydusitá chemie MeSH
- mitochondriální proteiny analýza metabolismus MeSH
- skot MeSH
- srdeční mitochondrie chemie metabolismus MeSH
- tyrosin analogy a deriváty analýza metabolismus MeSH
- zvířata MeSH
- Check Tag
- skot MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Heterologously expressed and purified azoreductase enzyme from facultative Klebsiella pneumoniae was used to degrade sulphonated azo dye. Methyl orange (MO) was used as the model dye to study the azo dye decolorization potential of the purified enzyme at different conditions. The enzyme had maximum activity at 40 °C and pH 8.0. The enzyme was observed to be thermo-stable as some enzyme activity was retained even at 80 °C. The apparent kinetic parameters, i.e., appKm and appVmax, for azoreductase using MO as a substrate were found to be 17.18 μM and 0.08/min, respectively. The purified enzyme was able to decolorize approximately 83% of MO (20 μM) within 10 min in the presence of NADH. Thus, efficient decolorization of MO was observed by the purified enzyme. The recombinant enzyme was purified approximately 18-fold with 46% yield at the end of four steps of the purification process. Enzyme was present in a tetrameric structure as confirmed by the volume at which protein was eluted in gel filtration chromatography, and the monomeric molecular mass of enzyme was found to be 23 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The dye degradation efficiency of azoreductase cloned from Klebsiella pneumoniae and purified from recombinant Escherichia coli was observed to be much higher as compared with the efficiencies of the reported azoreductases from other bacterial strains. In the present study, we report the purification and characterization of the azoreductase cloned from Klebsiella pneumoniae and expressed in Escherichia coli.
- MeSH
- azosloučeniny metabolismus MeSH
- bakteriální proteiny chemie genetika izolace a purifikace metabolismus MeSH
- barvicí látky metabolismus MeSH
- biodegradace MeSH
- Escherichia coli genetika metabolismus MeSH
- kinetika MeSH
- Klebsiella pneumoniae enzymologie genetika MeSH
- koncentrace vodíkových iontů MeSH
- molekulová hmotnost MeSH
- nitroreduktasy chemie genetika izolace a purifikace metabolismus MeSH
- rekombinantní proteiny chemie genetika izolace a purifikace metabolismus MeSH
- teplota MeSH
- Publikační typ
- časopisecké články MeSH
A specific type II restriction endonuclease T.Smu451I has been purified to electrophoretic homogeneity from the frozen cells of soil bacterium Sphingobacterium multivorum 451 (formerly Flavobacterium multivorum 451), using ultrasonic grinding, nucleic acid removal by streptomycin sulfate, protein precipitation by ammonium sulfate and phosphocellulose P-11, DEAE-Cellulose DE-52, Hepharin-Sepharose CL-6B chromatography, and elucidated several characteristics of T.Smu451I. The molecular weight of the enzyme determined by gel filtration and SDS-polyacrylamide gel electrophoresis was calculated to be 45,000 ± 2000 D (dimer) and 23,000 ± 1000 D (monomer), respectively. The isoelectric point (pI) of T.Smu451I is 5.4. T.Smu451I recognizes pentanucleotide palindromic sequences 5'-GGNC↓C-3' and cleaves between C and C in position shown by arrow to produce 3'-cohesive terminus of trinucleotide. Therefore, T.Smu451I is a neoschizomer of T.AsuI.
Thuricin 4AJ1, produced by Bacillus thuringiensis strain 4AJ1, showed inhibition activity against Bacillus cereus 0938 and ATCC 10987. It began to appear in the stationary phase and reached its maximum activity level of 209.958 U at 18 h against B. cereus 0938 and 285.689 U at 24 h against B. cereus ATCC 10987. Tricine-SDS-PAGE results showed that the partly purified thuricin 4AJ1 was about 6.5 kDa. The molecular weights of the known B. thuringiensis bacteriocins and the ones obtained by the two mainstream websites for predicting bacteriocins were inconsistent with the size of the thuricin 4AJ1, indicating that the bacteriocin obtained in this study may have a novel structure. Based on the biochemical properties, the thuricin 4AJ1 activities increased after treatment with proteinase K and lipase II, and were not affected by a-amylase, catalase, α-chymotrypsin VII and α-chymotrypsin II. It was heat tolerant, being active up to 90º C. In the pH 3-10 range, it maintained most of its activity. Finally, the sensitivity of the strain 4AJ1 to commonly used antibiotics was tested. In view of its stability and antibacterial activity, thuricin 4AJ1 may be applied as a food biopreservative.
- MeSH
- antibakteriální látky farmakologie MeSH
- Bacillus cereus účinky léků MeSH
- Bacillus thuringiensis chemie metabolismus MeSH
- bakteriociny chemie izolace a purifikace farmakologie MeSH
- elektroforéza v polyakrylamidovém gelu MeSH
- molekulová hmotnost MeSH
- potravinářská mikrobiologie MeSH
- Publikační typ
- časopisecké články MeSH
Many compounds have the potential to harm pancreatic beta-cells; organochlorine pollutants belong to those compounds. In this work, we aimed to find markers of acute toxicity of p,p'-DDT exposure among proteins expressed in NES2Y human pancreatic beta-cells employing 2-D electrophoresis. We exposed NES2Y cells to a high concentration (150 μM, LC96 after 72 hours) of p,p'-DDT for 24 and 30 hours and determined proteins with changed expression using 2-D electrophoresis. We have found 22 proteins that changed their expression. They included proteins involved in ER stress (GRP78, and endoplasmin), mitochondrial proteins (GRP75, ECHM, IDH3A, NDUS1, and NDUS3), proteins involved in the maintenance of the cell morphology (EFHD2, TCPA, NDRG1, and ezrin), and some other proteins (HNRPF, HNRH1, K2C8, vimentin, PBDC1, EF2, PCNA, biliverdin reductase, G3BP1, FRIL, and HSP27). The proteins we have identified may serve as indicators of p,p'-DDT toxicity in beta-cells in future studies, including long-term exposure to environmentally relevant concentrations.
- MeSH
- 2D gelová elektroforéza MeSH
- beta-buňky cytologie účinky léků metabolismus MeSH
- biologické markery metabolismus MeSH
- buněčné linie MeSH
- DDT toxicita MeSH
- hmotnostní spektrometrie MeSH
- lidé MeSH
- proteomika metody MeSH
- regulace genové exprese účinky léků MeSH
- viabilita buněk účinky léků MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
An attempt has been made to delineate the role of natural and synthetic retinoid receptor ligands on vimentin expression in the human triple-negative breast cancer cells. The effects of currently synthesized triorganotin derivatives of the general formula R3SnX (R is butyl or phenyl, X is isothiocyanate), which are considered RXR ligands, were investigated in the human MDA-MB-231 breast cancer cell line. Studies were evaluated in the presence and absence of all-trans retinoic acid (ATRA), a natural RAR ligand. Vimentin represents the major protein associated with epithelial-mesenchymal transition (EMT), an essential process when the primary tumour transforms into a malignant one. mRNA and proteomic data obtained in this study, based on the PDQuest software protein evaluation and further quantification of proteins by iTRAQ analysis, suggest that vimentin was significantly reduced in the combination of RAR ligand and RXR ligand treatment. Both tested triorganotin compounds showed similarly reduced expression of vimentin, but tributyltin isothiocyanate (TBT-ITC) proved to be more effective than triphenyltin isothiocyanate (TPT-ITC). Furthermore, the effect of natural (9cRA) and synthetic RXR ligands, both chloride and isothiocyanate derivatives, on vimentin expression was compared.
- MeSH
- 2D gelová elektroforéza MeSH
- antitumorózní látky farmakologie MeSH
- down regulace MeSH
- epitelo-mezenchymální tranzice účinky léků MeSH
- lidé MeSH
- nádorové buněčné linie MeSH
- nádory prsu farmakoterapie metabolismus patologie MeSH
- organocínové sloučeniny farmakologie MeSH
- proteomika metody MeSH
- retinoidní X receptory agonisté metabolismus MeSH
- signální transdukce účinky léků MeSH
- spektrometrie hmotnostní - ionizace laserem za účasti matrice MeSH
- tandemová hmotnostní spektrometrie MeSH
- tretinoin farmakologie MeSH
- trialkylcínové sloučeniny farmakologie MeSH
- vimentin metabolismus MeSH
- Check Tag
- lidé MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- srovnávací studie MeSH
