- In vitro reconstitution of yeast RNA polymerase II transcription initiation with high efficiency. [Journal Article]
- MMethods 2019 Mar 21
- Transcription initiation can be reconstituted from highly purified general transcription factors (GTFs), RNA polymerase II (pol II), and promoter DNA. However, earlier biochemical reconstitution syst…
Transcription initiation can be reconstituted from highly purified general transcription factors (GTFs), RNA polymerase II (pol II), and promoter DNA. However, earlier biochemical reconstitution systems had a serious technical limitation, namely very poor initiation efficiency. Due to the poor efficiency of the reaction and trace amounts of proteins involved in the pre-initiation complex (PIC) assembly, detection of transcription and PIC formation was only possible by the synthesis of a radiolabeled transcript and by immunoblotting for PIC components on templates. Here we describe a transcription system that is capable of initiating transcription with >90% efficiency of template usage using homogeneous, active yeast components including TFIIA, TFIIB, TBP, TFIIE, TFIIF, TFIIH, Sub1, and pol II. The abundant specifically assembled PICs on promoter DNA can be separated from free general transcription factors (GTFs) and pol II by density gradient sedimentation, irrespective of the length of promoter DNA. The system is robust, and can be modified to accommodate many other transcription factors, and the resulting complexes can be analyzed by SDS-PAGE followed by Coomassie Blue staining. This technical advance now paves the way to conduct definitive biochemical and structural studies of the complete process of pol II initiation from the PIC, through promoter escape, and finally to productive elongation.
- Analysis of Mitochondrial Respiratory Chain Complexes in Cultured Human Cells using Blue Native Polyacrylamide Gel Electrophoresis and Immunoblotting. [Journal Article]
- JVJ Vis Exp 2019 Feb 12; (144)
- Mitochondrial respiration is performed by oxidative phosphorylation (OXPHOS) complexes within mitochondria. Internal and environmental factors can perturb the assembly and stability of OXPHOS complex…
Mitochondrial respiration is performed by oxidative phosphorylation (OXPHOS) complexes within mitochondria. Internal and environmental factors can perturb the assembly and stability of OXPHOS complexes. This protocol describes the analysis of mitochondrial respiratory chain complexes by blue native polyacrylamide gel electrophoresis (BN-PAGE) in application to cultured human cells. First, mitochondria are extracted from the cells using digitonin, then using lauryl maltoside, the intact OXPHOS complexes are isolated from the mitochondrial membranes. The OXPHOS complexes are then resolved by gradient gel electrophoresis in the presence of the negatively charged dye, Coomassie blue, which prevents protein aggregation and ensures electrophoretic mobility of protein complexes towards the cathode. Finally, the OXPHOS complexes are detected by standard immunoblotting. Thus, BN-PAGE is a convenient and inexpensive technique that can be used to evaluate the assembly of entire OXPHOS complexes, in contrast to the basic SDS-PAGE allowing the study of only individual OXPHOS complex subunits.
- Beneficial effects of Coomassie staining on proteomic analysis employing PAGE separation followed with whole-gel slicing, in-gel digestion and quantitative LC-MS/MS. [Journal Article]
- JCJ Chromatogr B Analyt Technol Biomed Life Sci 2019 Mar 15; 1110-1111:25-35
- Proteomic analysis by combining PAGE separation, gel slicing and slice-by-slice in-gel digestion and LC-MS/MS has been frequently reported in recent years. Since the MS analysis would provide identit…
Proteomic analysis by combining PAGE separation, gel slicing and slice-by-slice in-gel digestion and LC-MS/MS has been frequently reported in recent years. Since the MS analysis would provide identities and quantities of the proteins along the whole lane, gel visualization by dye staining could be skipped to save time and labor. In this work, we examined the effects of CBB R-250 staining on the performance of the method and the results showed actually better results were obtained with CBB staining than without, both in nondenaturing PAGE and SDS-PAGE. A primary examination was firstly performed with excised gel bands of purified proteins and the LC-MS/MS results showed that almost all the proteins were detected with higher sequence coverages and quantities from the stained bands than from the unstained. Then a proteomic sample of rat heart soluble proteins was examined for the complete workflow. The sample was separated by both nondenaturing PAGE and SDS-PAGE and the gels were divided to halves for CBB staining and fixation without staining, respectively. Multi-blade cutters were used to simultaneously cut lanes and then slice each lane into about forty squares of the same size. All the gel pieces were analyzed in standard procedures of in-gel digestion, peptide extraction and label-free quantitative LC-MS/MS. The results showed more proteins, about 40% in nondenaturing PAGE and 18% in SDS-PAGE, were detected from the CBB-stained lanes than from the unstained ones. Examination on the detected quantities and square numbers of individual proteins also confirmed about the better detection with CBB staining. As the data showed the detection of proteins with lower molecular masses (e.g. <30 kDa) were more benefited by the staining, we speculate the dye binding might help retaining of the proteins in the gel matrix.
- Histological and immunohistochemical characterization of the integument and parotoids glands Rhinella bergi (Anura: Bufsonidae): Development and differentiation. [Journal Article]
- AHActa Histochem 2019; 121(3):277-283
- A detailed description of the tegument and parotoid glands of pre-metamorphic, post-metamorphic, juvenile and adult individuals of Rhinella bergi is presented to provide an exhaustive analysis of the…
A detailed description of the tegument and parotoid glands of pre-metamorphic, post-metamorphic, juvenile and adult individuals of Rhinella bergi is presented to provide an exhaustive analysis of the integumentary characteristics of this species. Fragments of the tegument were fixed in Bouin solution and preserved in buffered Formol 10%. Subsequently, scanning electron microscopy (SEM) was performed to characterize the macroscopic structure of these regions. Microscopic observations were made from histological sections stained with Hematoxylin and Eosin, Alcian Blue (pH 2,5), PAS-H, Coomassie Blue, Oil Red, and Bielschowsky Impregnation.. There were three types of protuberance: warts, tubers, and thorns. These structures became evident from post-metamorphic stages. The ventral surface shows elevations similar to flat warts; however, tubers and spines are absent. Histologically, each structure consists of a spongy dermis of lax connective tissue and dense and compact dermis, associated with granular glands and a keratinized epidermis. The latter, in the dorsal region, forms projections called thorns. The granular glands accumulate, and their alveoli increase in size progressively. This work provides a morphological and histological description of the integument and the parotoid glands during the larval and post-metamorphic stage of the genus Rhinella, with aspects described for the first time in the genus.
- In Vitro Cytotoxicity Evaluation of the Magnéli Phase Titanium Suboxides (TixO2x-1) on A549 Human Lung Cells. [Journal Article]
- IJInt J Mol Sci 2019 Jan 08; 20(1)
- The use of titanium suboxides, known as Magnéli phase TiOx, is expected to increase in the near future due to their desirable properties. In order to use Magnéli phase TiOx nanoparticles safely, it i…
The use of titanium suboxides, known as Magnéli phase TiOx, is expected to increase in the near future due to their desirable properties. In order to use Magnéli phase TiOx nanoparticles safely, it is necessary to know how nanoparticles interact with biological systems. In this study, the cytotoxicity of three different Magnéli TiOx nanoparticles was evaluated using human lung A549 cells and the results were compared with hazard data on two different TiO₂ nanoparticles whose biological interactions have already been extensively studied. After A549 cells were exposed to nanoparticles, the metabolic activity was measured by the Resazurin assay, the amount of cellular proteins was measured by the Coomassie Blue assay, and lysosomal integrity was measured by the Neutral Red Uptake assay. In order to investigate possible modes of particle actions, intracellular Ca2+ level, reactive oxygen species (ROS) production, and photo-oxidative disruptions of lysosomal membranes were assessed. All experiments were performed in serum-containing and in serum-deprived cell culture mediums. In addition, the photocatalytic activity of Magnéli TiOx and TiO₂ nanoparticles was measured. The results show that Magnéli TiOx nanoparticles increase intracellular Ca2+ but not ROS levels. In contrast, TiO₂ nanoparticles increase ROS levels, resulting in a higher cytotoxicity. Although Magnéli TiOx nanoparticles showed a lower UV-A photocatalytic activity, the photo-stability of the lysosomal membranes was decreased by a greater extent, possibly due to particle accumulation inside lysosomes. We provide evidence that Magnéli TiOx nanoparticles have lower overall biological activity when compared with the two TiO₂ formulations. However, some unique cellular interactions were detected and should be further studied in line with possible Magnéli TiOx application. We conclude that Magnéli phase nanoparticles could be considered as low toxic material same as other forms of titanium oxide particles.
- [Aliskiren inhibits angiotensin II/angiotensin 1-7(Ang II/Ang1-7) signal pathway in rats with diabetic nephropathy]. [Journal Article]
- XBXi Bao Yu Fen Zi Mian Yi Xue Za Zhi 2018; 34(10):891-895
- Objective To investigate the role of aliskiren (AL) in the angiotensinII/ angiotensin 1-7 (AngII/Ang1-7) signal pathway in renal tissue of diabetic nephropathy (DN) rats. Methods 40 male SD rats were…
Objective To investigate the role of aliskiren (AL) in the angiotensinII/ angiotensin 1-7 (AngII/Ang1-7) signal pathway in renal tissue of diabetic nephropathy (DN) rats. Methods 40 male SD rats were randomly divided into 4 groups including normal group, diabetes group, AL group and valsartan (Val) group. Animal models of diabetes were induced by high fat diet combined with small dose of streptozotocin injection. Rats in AL group were administered 50 mg/(kg.d) AL by gavage, and rats in Val group were administered 30 mg/(kg.d)Val by gavage. 24-hour urine protein (24 h-UP) were observed by Coomassie blue colorimetry at 2, 4 and 6 weeks after modeling, serum creatinine was detected by automatic biochemical analyser, kidney index [kidneys mass(g)/body mass (kg)] was measured. HE and PAS staining were used to observe renal pathological changes. Immunohistochemistry was used to detect the expression of ACE2, MAS receptor, AT1R and AT2R in the kidney. Results After 6 weeks of modeling, there was no significant difference in creatinine level among groups. The levels of glucose, 24 h-UP and kidney index in diabetes group, AL group and Val group significantly increased. Compared with diabetes group, the levels of 24 h-UP and kidney index were lower in AL group and Val group. Immunohistochemistry showed that the expression of AT2R and ACE2 was lower and the expression of MAS receptor was higher in AL group than diabetes group and Val group. Compared to normal group and Val group, AT1R expression was significantly up-regulated in AL group, without significant difference between diabetes group and AL group. Conclusion AL down-regulates the expression of AT2R and ACE2, thus inhibits the AngII/ Ang1-7 signal axis and improves/alleviates the symptoms in diabetic rats.
- Quantitative measurement of urinary proteins in 2018: advantages, disadvantages, limits. [Journal Article]
- ABAnn Biol Clin (Paris) 2018 Dec 01; 76(6):627-631
- Today, there is no reference method for the measurement of urinary proteins. The difficulties are that urine is a very complex biological fluid, and that there are a high intra-and inter-individual v…
Today, there is no reference method for the measurement of urinary proteins. The difficulties are that urine is a very complex biological fluid, and that there are a high intra-and inter-individual variability in the protein excretion rate. Progress has been made during the last thirty years, but high analytical variability persists among the colorimetric or turbidimetric methods used for urinary proteins measurement.
- Crystal violet stains proteins in SDS-PAGE gels and zymograms. [Journal Article]
- ABAnal Biochem 2019 Feb 01; 566:107-115
- Coomassie brilliant blue R250, an anionic dye is the most popular stain to detect proteins resolved in SDS-PAGE gels. Crystal violet, a cationic dye was found to be versatile and stained proteins in …
Coomassie brilliant blue R250, an anionic dye is the most popular stain to detect proteins resolved in SDS-PAGE gels. Crystal violet, a cationic dye was found to be versatile and stained proteins in SDS-PAGE gels and in zymograms. Stained proteins can be transferred to nitrocellulose and the stained proteins on the western detected with enzyme coupled antibodies. Staining can be reversed. Staining takes 3 h at RT or 30 min at 60 °C. Crystal violet stained some E. coli high and low molecular weight proteins not stained by Coomassie blue R250. Crystal violet stained down to 16 ng of protein, some five-fold lower than Coomassie blue, though the two stains had a similar linear dynamic range. The staining sensitivity could be increased to 2 ng when crystal violet and Coomassie blue were combined in a double staining/counterion dye formulation. The low concentrations of the dye without a destaining step reduces the costs of the technique and results in a more environmentally friendly stain compared to traditional staining methods.
- Measuring Protein Concentration with Absorbance, Lowry, Bradford Coomassie Blue, or the Smith Bicinchoninic Acid Assay Before Electrophoresis. [Journal Article]
- MMMethods Mol Biol 2019; 1855:31-39
- Measuring the concentration of proteins is an essential part of enzyme analysis or serves to monitor protein yields and losses during protein isolation procedures. Decisions on the usefulness of any …
Measuring the concentration of proteins is an essential part of enzyme analysis or serves to monitor protein yields and losses during protein isolation procedures. Decisions on the usefulness of any protein isolation procedure depend on knowing the concentration of proteins before and after a procedure. Protein concentration in solution is generally measured with spectrophotometry in the UV range or in the presence of dyes or copper interacting with the protein. This review describes absorbance at 280 nm, the Lowry, Bradford (Coomassie Blue), and Smith (bicinchoninic acid) assays for measuring protein and includes suggestions for optimizing each method.
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- The effects of IAM38 blocking or CD4 blocking on the binding of exogenous DNA in rabbit sperm. [Journal Article]
- MBMol Biol Rep 2019; 46(1):251-259
- The binding of exogenous DNA to sperm is a key process for sperm-mediated gene transfer; however, the underlying molecular mechanisms have yet to be elucidated. In the present study, we aimed to iden…
The binding of exogenous DNA to sperm is a key process for sperm-mediated gene transfer; however, the underlying molecular mechanisms have yet to be elucidated. In the present study, we aimed to identify the DNA binding proteins (DBPs) in rabbit sperm and to gain further understanding of the molecular mechanism of sperm and exogenous DNA interaction. Native polyacrylamide gel electrophoresis was used for separating free sperm proteins and complexes of DNA fragment/sperm proteins. A distinct band was found after Coomassie blue staining, and seven potential proteins were identified by mass spectrometry analysis. An analysis of the physical/chemical properties of the seven proteins revealed that the sperm inner acrosomal membrane protein IAM38 (IAM38) matched the features of the DBPs. Western blotting analysis showed that the IAM38 and CD4 were present in the sperm but not in the seminal plasma. Blocking of the IAM38 impaired the DNA-binding capacity of the sperm. Blocking the CD4 decreased the DNA-uptake capacity of the sperm but did not influence the DNA-binding capacity of the sperm. Moreover, the EGFP-positive embryos and EGFP-positive blastocysts were also decreased after IAM38 blocking or CD4 blocking in comparison with the control group. In conclusion, our results imply that foreign DNA first binds to the transmembrane IAM38 of the sperm plasma membrane and then forms the complex of DNA/IAM38/CD4 with CD4 to complete the transportation of exogenous DNA into the nucleus of sperm.