OBJECTIVE: To evaluate the presence and activity of local sperm antibodies and electrophoretic analysis of ovulatory cervical mucus (OCM) to prove the correlation of results of Kremer mucus sperm capillary penetration test and i-MAR (mixed antiimmunoglobulin reaction) test with SDS-PAGE. DESIGN: Retrospective study. SETTING: Department of Obstetrics and Gynecology, Medical Faculty of Charles University, Pilsen. METHODS: 94 patients aged 22-40 (average age 32.3 years) were chosen for our study. Ovulatory cervical mucus was taken from uterine cervical canal in Consultation for Reproductive Immunology. Kremer test and indirect mixed antiglobulin reaction test for IgG, IgA, IgM and IgE were used for detection of sperm antibodies. For the SDS-PAGE analysis, OCM was incubated with sodium dodecyl sulfate. We studied separated protein fractions from OCM. RESULTS: Sperm-capillary ovulatory mucus penetration test (Kremer) was 0-10 cm/hour in our group (average value in patiens without sperm antibodies was 2.43 cm/hour, with sperm agglutinating antibodies 1.4 cm/hour), significant levels (> 45%) of spermagglutinating antibodies were detected in IgA in 6 patients (6.38%), IgG in 5 (5.32%) patients, sperm-cytotoxic levels (IgA and/or IgG) in 5 patients (5.32%); levels of IgE less than 30% in 3 patients (3.19%). Individual immunological factors gained by SDS-PAGE showed the spectrum of various molecular weights with range of 14.4- 350 kDa. The presence of IgG and/or IgA was in 53 cases (56.38%), with 13 ASA positivities (24.53% correlation with i-MAR test) and no ASA activity in 40 cases (75.47%); 6 ASA positive patiens (31.58%) were not detected by SDS-PAGE. CONCLUSIONS: Analysis of 94 OCM by SDS-PAGE showed several significant correlates, but their specifications will be based on further immunoblot research.

• MeSH
• aglutinace spermií MeSH
• cervikální hlen imunologie   MeSH
• dospělí MeSH
• elektroforéza v polyakrylamidovém gelu MeSH
• fertilizace in vitro * MeSH
• imunoglobuliny analýza   MeSH
• lidé MeSH
• neúspěšná terapie MeSH
• ovulace * MeSH
• protilátky analýza   MeSH
• spermie imunologie   MeSH
• ženská infertilita imunologie   terapie   MeSH
• Check Tag
• dospělí MeSH
• lidé MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• Publikační typ
• anglický abstrakt MeSH
• časopisecké články MeSH
• Názvy látek
• imunoglobuliny MeSH
• protilátky MeSH

The study of membrane proteins and membrane protein complexes (MPC) provides crucial information in the field of bacterial physiology and pathogenesis research. The method of blue native polyacrylamide gel electrophoresis and its combination with SDS-PAGE (BN/SDS-PAGE) were here employed to study the membrane complexome of an intracellular bacterium Francisella tularensis, the causative agent of a severe disease tularemia. In the presented study we describe the subunit composition and stoichiometry of several MPC involved in various cell functions (oxidative phosphorylation, membrane transport, cell division, membrane or periplasmic proteins folding, iron storage, phospholipid and cell envelope biosynthesis). Moreover, some undocumented or hypothetical MPC with possible connection to virulence factors were also proposed and some newly detected subunits were assigned to known complexes. The BN/SDS-PAGE combined with mass spectrometry appeared to be a strong tool in the investigation of membrane proteins and complexes and thus successfully complements the traditional electrophoresis approaches.

• MeSH
• barvicí látky chemie   MeSH
• elektroforéza v polyakrylamidovém gelu metody   MeSH
• Francisella tularensis metabolismus   MeSH
• lidé MeSH
• membránové proteiny analýza   chemie   metabolismus   MeSH
• molekulová hmotnost MeSH
• multiproteinové komplexy analýza   chemie   metabolismus   MeSH
• tularemie metabolismus   patologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• barvicí látky MeSH
• membránové proteiny MeSH
• multiproteinové komplexy MeSH

The time required to visualize proteins using Coomassie Blue dye has been significantly reduced with the introduction of fast staining protocols based on staining with a Coomassie Blue dye solution at boiling temperatures. However, fast stainings suffer from high gel backgrounds, reducing the signal-to-noise ratio and limiting the number of detectable spots in the case of 2D SDS-PAGE. The aim of this work was to eliminate the high gel background, and thus improve fast staining protocols based on Coomassie Blue dye. We show that merely replacing water with a 4 mM EDTA washing solution at boiling temperatures, results in a transparent gel background within 50 to 60 minutes of destaining. Moreover, when a combination of imidazole-zinc reverse staining and Coomassie Blue-based fast staining is used the sensitivity is improved significantly; nanogram amounts of proteins can be detected using 1D SDS-PAGE, and about 30% to 60% more spots can be detected with 2D SDS-PAGE in plasma, platelet, and rat brain tissue samples. This work represents an optimized fast staining protocol with improved sensitivity, requiring between 60 to 75 minutes to complete protein visualization.

• MeSH
• elektroforéza v polyakrylamidovém gelu metody   MeSH
• indikátory a reagencie chemie   MeSH
• proteiny izolace a purifikace   MeSH
• rosanilinová barviva chemie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• Coomassie blue MeSH Prohlížeč
• indikátory a reagencie MeSH
• proteiny MeSH
• rosanilinová barviva MeSH

PURPOSE: The goal of this study was to design an easy and simple protocol for platelet isolation and sample preparation for proteomic studies based on 2DE (IEF-SDS-PAGE) followed by Coomassie blue staining. EXPERIMENTAL DESIGN: Blood was collected by venipuncture into tubes coated with EDTA and platelet-rich plasma (PRP) was immediately obtained by centrifugation. PRP was stored refrigerated in closed Falcon tubes for 0, 1, 2, 3, 5, and 7 days and platelets were isolated by centrifugation. 2DE gels were stained with colloidal Coomassie blue stain and evaluated using the Progenesis SameSpots software. Spots that differed significantly in the gels of fresh and stored platelet samples were excised, digested with trypsin, and further analyzed using nanoLC-MS/MS. RESULTS: During the 7-day follow-up period, we found 20 spots that differed significantly (ANOVA p <0.05). During the first 2 days of PRP storage in test tubes, however, only nine spots significantly differed in all donors. In these spots, we identified 14 different proteins. CONCLUSIONS AND CLINICAL RELEVANCE: In conclusion, for proteome investigations, whenever it is not feasible to prepare washed platelets immediately after blood collection, the EDTA-anticoagulated PRP can be stored in test tubes at 4°C for up to 2 days for the platelet proteome investigation.

• MeSH
• 2D gelová elektroforéza MeSH
• agregace trombocytů účinky léků   MeSH
• elektroforéza v polyakrylamidovém gelu metody   MeSH
• kyselina arachidonová farmakologie   MeSH
• lidé MeSH
• metody pro přípravu analytických vzorků * MeSH
• plazma bohatá na destičky metabolismus   MeSH
• proteomika metody   MeSH
• receptory trombinu metabolismus   MeSH
• trombocyty metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• kyselina arachidonová MeSH
• receptory trombinu MeSH

Protein phosphorylation is the most abundant and best studied protein posttranslational modification, dedicated to the regulation of protein function and subcellular localization as well as to protein-protein interactions. Identification and quantitation of the dynamic, conditional protein phosphorylation can be achieved by either metabolic labeling of the protein of interest with (32)P-labeled ATP followed by autoradiographic analysis, the use of specific monoclonal or polyclonal antibodies against the phosphorylated protein species and finally by phosphoproteome delineation using mass spectrometry.Hereby we present a fourth alternative which relies on the enforced-affinity-based-electrophoretic separation of phosphorylated from non-phosphorylated protein species by standard SDS-PAGE systems co-polymerized with Phos-Tag™ and Mn(2+) or Zn(2+) cations. Phosphate groups of phosphorylated Ser, Thr, and Tyr residues form complexes with Mn(2+) and Zn(2+) cations with polyacrylamide immobilized Phos-Tag™. Following appropriate treatment of the gels, separated proteins can be quantitatively transferred to PVDF or nitrocellulose membranes and probed with common-not phosphorylation state specific-antibodies and delineate the occurrence of a certain phosphoprotein species against its non-phosphorylated counterpart.

• MeSH
• akrylamid chemie   MeSH
• Arabidopsis enzymologie   růst a vývoj   MeSH
• bakteriofág lambda enzymologie   MeSH
• elektroforéza v polyakrylamidovém gelu metody   MeSH
• fenol chemie   MeSH
• fosfatasy metabolismus   MeSH
• fosfoproteiny izolace a purifikace   metabolismus   MeSH
• fosforylace MeSH
• kultivační techniky MeSH
• membrány umělé MeSH
• mitogenem aktivované proteinkinasy izolace a purifikace   metabolismus   MeSH
• polyvinyly chemie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• akrylamid MeSH
• fenol MeSH
• fosfatasy MeSH
• fosfoproteiny MeSH
• membrány umělé MeSH
• mitogenem aktivované proteinkinasy MeSH
• polyvinylidene fluoride MeSH Prohlížeč
• polyvinyly MeSH

Fast-staining protocols based on the use of Coomassie blue dye for SDS-PAGE separated proteins, represent a quick and simple solution for protein visualization. It has been shown however, that in some cases a phenomenon of missing spots or spot discoloration may be observed in the proteome pattern when the standard fast-staining protocol is used. In this work, it is demonstrated that this occurrence is affected by the biological variability of samples, and therefore, cannot be observed in all samples. Moreover, it is demonstrated that the phenomenon is manifested exclusively in nonfixed gels, and that including a fixation step into the fast-staining protocol prevented this phenomenon. In conclusion, it has been demonstrated that standard Coomassie blue dye based fast staining for SDS-PAGE resolved proteins is affected by the biological variability of samples in nonfixed gels.

• Klíčová slova
• Coomassie, Fast staining, Fixing, Imidazole-zinc, Missing spot,
• MeSH
• 2D gelová elektroforéza MeSH
• elektroforéza v polyakrylamidovém gelu metody   MeSH
• imidazoly MeSH
• proteiny analýza   chemie   MeSH
• reprodukovatelnost výsledků MeSH
• rosanilinová barviva chemie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• Coomassie blue MeSH Prohlížeč
• imidazole MeSH Prohlížeč
• imidazoly MeSH
• proteiny MeSH
• rosanilinová barviva MeSH

We developed a new method for the quantitative determination of myosin heavy chain (MyHC) isoforms taking advantage of immunochemical differences and based on the ELISA principle. In the present paper we compare analysis of MyHC isoforms using the SDS-PAGE and the ELISA methods in the same samples of adult female inbred Lewis strain euthyroid, hyperthyroid and hypothyroid rats. In all thyroid states, the same composition and corresponding changes of MyHC isoforms were determined using both methodological approaches in the slow soleus and the fast extensor digitorum longus muscles. Our results showed that ELISA can be used for a "semi-quantitative" or "comparative" measurement of MyHC isoforms in multiple muscle samples, but that it is neither more exact nor faster compared to SDS-PAGE.

• MeSH
• elektroforéza v polyakrylamidovém gelu MeSH
• ELISA MeSH
• inbrední kmeny potkanů MeSH
• krysa rodu Rattus MeSH
• potkani inbrední LEW MeSH
• protein - isoformy analýza   MeSH
• svalová vlákna typu I chemie   fyziologie   MeSH
• svalová vlákna typu II chemie   fyziologie   MeSH
• těžké řetězce myosinu analýza   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• srovnávací studie MeSH
• Názvy látek
• protein - isoformy MeSH
• těžké řetězce myosinu MeSH

Proteomic-based description of varieties of barley (Hordeum vulgare L.) is a very important task especially in the food and brewing industry. This study is focused on major storage proteins in barley--hordeins--as a group of proteins soluble in alcohol/water mixtures that shows up significant changes in proteomic profiles for different varieties of barley. Unusual amino acid composition of hordeins with low numbers of lysine and arginine in combination with high content of proline and glutamine complicates their identification in a common proteomic workflow, because tryptic digestion produces just a few peptides amenable for successful mass spectrometric analysis. To increase the number of cleavage sites, in this work, cysteines in hordeins were chemically modified with 2-bromoethylamine (BEA) for their conversion into aminoethylcysteines. These mimic lysine residues and are recognized by trypsin as potential cleavage sites (if not followed by a proline residue) on the C-terminal side of the modified cysteine. Small extent of side reactions (towards histidine, N-terminus of the peptide, methionine, and also here the newly discovered reaction towards aminoethylcysteine) during modification with BEA could be observed after a longer period of reaction but this did not hinder the analysis when optimal conditions were used. Application of trypsin for in-gel digestion of hordeins, previously modified chemically with BEA, provided a higher number of short peptides and their subsequent mass spectrometric analysis resulted in an improved identification of hordeins. This approach can also be used for the analysis of other similar protein groups (e.g. gliadins in wheat) or other cysteines containing proteins having a low number of lysine and arginine residues in their primary structure.

• MeSH
• alkylace MeSH
• cystein chemie   metabolismus   MeSH
• elektroforéza v polyakrylamidovém gelu metody   MeSH
• ethylaminy chemie   MeSH
• gluteny chemie   metabolismus   MeSH
• ječmen (rod) chemie   MeSH
• peptidové fragmenty MeSH
• trypsin metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• 2-bromoethylamine MeSH Prohlížeč
• cystein MeSH
• ethylaminy MeSH
• gluteny MeSH
• peptidové fragmenty MeSH
• trypsin MeSH

The methicillin-resistant Staphylococcus aureus causes difficult-to-treat healthcare-associated infections in humans. For fast and effective selection of an appropriate antibiotic therapy, it is essential to have rapid and reliable methods for differentiation of methicillin-resistant S. aureus from less dangerous methicillin-sensitive S. aureus. There have been many methods for the identification of methicillin-resistant S. aureus described but none has been accepted as an international standard. The most commonly used techniques such as phenotyping and genotyping have a few disadvantages, for instance, these techniques are not reproducible and stable. In addition, they are time-consuming, expensive, and they are not capable to distinguish all S. aureus strains. In this study, the methicillin-resistant and methicillin-sensitive S. aureus isolates obtained from patients were extracted in hot water. The released proteins were characterised by sodium dodecyl sulphate polyacrylamide gel electrophoresis and gel isoelectric focusing. These two methods were able to differentiate among tested bacterial strains. The proposed methods are time saving, they are applicable in standard biochemical laboratories, and they do not require any expensive equipment.

• MeSH
• bakteriologické techniky metody   MeSH
• časové faktory MeSH
• elektroforéza v polyakrylamidovém gelu metody   MeSH
• isoelektrická fokusace metody   MeSH
• lidé MeSH
• rezistence na methicilin * MeSH
• Staphylococcus aureus chemie   klasifikace   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• hodnotící studie MeSH
• práce podpořená grantem MeSH

The relationship between gushing and antifungal peptides in barley and malt kernels was examined for five barley varieties produced in the Czech Republic with four conditions of infection and treatment. Proteome changes during pathogen-seed interaction were observed with SDS-PAGE and MALDI-TOF MS. These methods were applied as a fast screening for observing the relationship between gushing and peptides/proteins. It was found that the presence of basic peptides, presumably hordothionins and non-specific lipid transfer protein type 1, did not correlate with the degree of gushing for malt (/r/ in <0.07, 0.34>), (/r/ in <0.01, 0.49>), respectively, as detected by both methods.

• MeSH
• antifungální látky analýza   MeSH
• elektroforéza v polyakrylamidovém gelu MeSH
• houby patogenita   MeSH
• ječmen (rod) chemie   mikrobiologie   MeSH
• kationické antimikrobiální peptidy analýza   MeSH
• pivo analýza   mikrobiologie   MeSH
• proteiny teplotního šoku analýza   MeSH
• proteiny vázající mastné kyseliny MeSH
• rostlinné proteiny ve výživě analýza   MeSH
• rostlinné proteiny analýza   MeSH
• spektrometrie hmotnostní - ionizace laserem za účasti matrice MeSH
• transportní proteiny analýza   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Geografické názvy
• Česká republika MeSH
• Názvy látek
• antifungální látky MeSH
• hordothionin protein, Hordeum vulgare MeSH Prohlížeč
• kationické antimikrobiální peptidy MeSH
• Ltp1 protein, barley MeSH Prohlížeč
• proteiny teplotního šoku MeSH
• proteiny vázající mastné kyseliny MeSH
• rostlinné proteiny ve výživě MeSH
• rostlinné proteiny MeSH
• transportní proteiny MeSH