Distribution of Coomassie Blue Stainable Particles in the Pearl River Estuary, China, Insight Into the Nitrogen Cycling in Estuarine System

Distributions of Coomassie Blue stainable particles (CSP), the sources and transports, as well as their implications for nitrogen biogeochemical cycles in the Pearl River estuary (PRE) were investigated during two cruises in August 2016 and January 2017. CSPcolor concentrations (CSP concentration determined spectrophotometrically) were 73.7–685.3 μg BSA eq L–1 [μg Bovine serum albumin (BSA) equivalent liter–1] in August 2016 and 100.6–396.4 μg BSA eq L–1 in January 2017, respectively. CSP concentrations were high in low-salinity waters (<5), and declined from the river to the middle estuary by 80% in the wet season and 55.6% in the dry season, respectively, then increased again in the lower estuary due to high primary production. CSP concentrations were mainly associated with chlorophyll a (Chl a) concentration except for the turbid mixing zone, suggesting that autochthonous phytoplankton production served as the primary source of CSP in the PRE. The concentrations of nitrogen (N) as CSP in the PRE were comparable to the nitrogen content of particulate hydrolysable amino acids (PHAA). Pictures of CSP taken by microscopy and the correlation between composition of PHAA and the ratio of Chl a/CSPcolor showed that CSP were relatively degraded due to delivery of old terrestrial protein to river section and extensive microbial degradation during mixing at the upper and middle parts of the estuary, whereas CSP in lower estuary appeared to be more labile due to higher fresh algal production. The contribution of CSP nitrogen (CSP-N) to the particulate nitrogen (PN) pool was 34.98% in summer and 30.8% in winter. The conservative estimate of CSP-N input flux in the Pearl River Delta was about 6 × 106 mol N d–1. These results suggested that CSP was a significant pool of organic nitrogen in the PRE. The study of CSP composition in terms of nitrogen provides new insight into the roles of CSP on nitrogen biogeochemical processes in the turbid and productive estuarine system.

Equine synovial fluid protein equalization via combinatorial peptide ligand libraries

To gain further knowledge of the equine synovial fluid (SF) proteome, we propose a protocol based on the equalization of the relative concentrations of its proteins, which leads to the modification of the standard electrophoretic pattern revealing low-abundance proteins that otherwise remain undetected. Fresh SF samples were collected from ten macroscopically normal metacarpophalangeal joints of crossbred horses. The samples were processed using standard procedures as the control and via combinatorial peptide ligand libraries (CPLL) using low ionic forces (NaH2PO4 10 mM) at different pHs (4.0, 7.0, and 9.3) with 10% sodium dodecyl sulfate (SDS) and 25 mM DTT for protein resolubilization. Proteins were then separated by conventional 8% SDS-PAGE and stained with coomassie blue. After separation of the equalized proteins, there was a significant reduction in the albumin (the most abundant protein in the SF) and, at the same time, other protein bands arise that were not visible without CPLL processing. In addition, there was variation in the protein profiles at different pHs. The results suggest that protein equalization of the equine SF by CPLL could be a useful tool to better understand the articular homeostasis and/or for the detection of new biomarkers of joint pathology.

SDS-PAGE gel electrophoresis v2

SDS-PAGE gel electrophoresis protocol for analyzing samples from plant leaf tissue via immunofluorescence. In this protocol no Coomassie blue is added to samples, the reason is that this interferes with the fluorescent signal during immunoblot. Instead, samples have already been prepared in Laemmli buffer (minus coomassie, protein extraction procedure), the leading edge of samples can be visualized due to the presence of chlorophyll. Note - When using 15 well, 0.75 mm comb, try to limit the volume loaded to 10 μL to minimize the risk of spillover of protein between wells. - Ensure that accurate volume is pipetted by removing sample stuck to the outside of the pipette tip by wiping the tip on the rim of the sample tube to remove any residual liquid. Literature: http://www.bio-rad.com/webroot/web/pdf/lsr/literature/Bulletin_6040.pdfhttps://www.bio-rad.com/webroot/web/pdf/lsr/literature/10026447.pdf

Antigen Similarity In Hydatid Cyst Wall And Human Bone Tumours: A Short Report

Background: less studies have been done on bone cancers which are complex despite lower incidance. Hydatidosis is a parasitic disease that may influence host immunity by mimicking cancer cells antigens. So, this study aimed to elavuate the similarity of the immunogenic antigens between hydatid cyst and different bone cancers.Method: Cyst wall of hydatid cysts were collected and their antigens were separated with SDS-PAGE gel electrophoresis (SDS-PAGE). Serum samples obtained from patients with bone cancers and the anitigenicity of isolated anitgens were evaluated inwith E. granulosus( Larval form )infection and healthy individuals using western-blot approaches. Results: The crude extract of the laminated layer showed two specific antigens, 53 KDa and 70 KDa, after stainging the membrane with Coomassie blue. Both antigens reacted with the serum of different bone cancers but only the 53 KDa band reacted with all sera.Conclusion: It seems people with bone tumours may have extra antibodies in their serum comparing to healthy and hydatidosis which may be an autoantibodies; and the presence of this antibody against 70 KDa band protein in sera of patients with various types of bone cancers, may be helpful in diagnostic test or designing of preventive approaches in future.

A comparative study of two methods to determine acrosome integrity of frozen-thawed boar sperm

Coomassie blue staining has been reported as an effective and inexpensive method for evaluating the acrosome integrity of spermatozoa, though to date its use to evaluate cryopreserved boar sperm has not been reported. Moreover, there is no information concerning the agreement between Coomassie blue staining and fluorescein isothiocyanate conjugated peanut agglutinin and ethidium homodimer (FITC-PNA/EthD-1) methods for assessing sperm acrosome integrity for any species. The current study was performed to determine the efficacy and agreement between Coomassie blue and FITC-PNA/EthD-1 staining methods for evaluating the acrosome integrity of frozen-thawed boar sperm. A total of 25 semen samples were cryopreserved using lactose-egg yolk-based extender and loaded into 0.5 PVC-French straws. Sperm motility and motion characteristics were determined using a computer-assisted sperm analysis system. Sperm viability and plasma membrane integrity were evaluated using the SYBR-14/EthD-1 and hypo-osmotic swelling test, respectively. Acrosome integrity of frozen-thawed boar sperm was evaluated using both FITC-PNA/EthD-1 and Coomassie blue staining to assess the association between sperm acrosome integrity and agreement between these two methods. The average percent acrosome integrity of frozen-thawed boar sperm as determined by FITC-PNA/ EthD-1 and Coomassie blue staining was 48.8 ± 12.6% and 52.6 ± 13.6%, respectively (P>0.05). Interestingly, Coomassie blue staining found a correlation between sperm viability and acrosome integrity (r=0.609, P=0.002), while FITC-PNA/EthD-1 staining did not (P>0.05). However, the acrosome integrity of frozen-thawed boar sperm evaluated by FITC-PNA/ EthD-1 and Coomassie blue staining was significantly correlated (r=0.448, P=0.025, n=25). The Bland-Altman plot determined that this agreement was acceptable. In conclusion, the acrosome integrity of the frozen-thawed boar sperm assessed via Coomassie blue staining was significantly correlated with that obtained via the FITC-PNA/EthD-1 staining method, and the two methods showed good agreement. Moreover, the significant association between the acrosome integrity of frozen-thawed boar sperm determined by Coomassie blue staining with other sperm quality parameters indicates that this is an effective method for assessing the acrosome integrity of frozen-thawed sperm in pigs.

Optimization of protein extraction and ELISA immunodetection from protein-based paint models with mesoporous silica nanoparticles and MCM41

Protein-based biological materials such as albumin, casein and collagen are found in various cultural heritage (CH) artefacts. This study focuses on the study of protein binders from easel paintings media. Proteins have complex structures which are difficult to identify with non-invasive spectroscopic methods (FT-IR, Raman, UV). Immunoassays such as ELISA determine the protein’s source of origin which is necessary for art objects. To increase the detection and identification of proteins by immunoassays, the efficiency of micro-extraction of proteins from heritage materials is a crucial step. Extractions mediated by cycles of orbital agitation and ultrasonic radiation give the possibility to extract proteins from easel painting sample. In this work, protein-based paint models coupled with silica nanoparticles were used for micro-extraction. Nanoparticles possess high surface-to-volume ratios that can attach bioactive molecules such as proteins and increase the total protein recovered from microsamples. Protein extracts were quantified with Bradford Assay in the presence of Coomassie blue. The protein recovery results were statistically computed, and the SPSS analysis shows significant (p < 0.05) increase in protein recovery, above 1.3 times for NPSiO2 and above 1.6 times for MCM-41. The statistical data shows evidence that silica nanoparticles intensify the total protein recovered from paint microsamples. Finally, ELISA was realized on the protein extracts to verify and compare the immunodetection of protein from the paint models with and without the use of silica nanoparticles.

A rapid method for protein staining in polyacrylamide gels using water saturated with chloroform

Polyacrylamide gel electrophoresis, followed by an appropriate staining, is a popular and useful analytical procedure for protein identification and characterization. The aim of this study was to develop a method for protein visualization in polyacrylamide gels that would be alternative to Coomassie blue or silver staining. The proposed method is simple, fast and inexpensive. The optimized protocol for protein staining and visualization takes as little as 6 minutes and utilizes deionized water and chloroform. Fluorescence of proteins is induced by UV light and can be detected with a standard transilluminator.

Protein Extractions from Amphistegina lobifera: Protocol Development and Optimization

Proteins are essential to life, and the evaluation of their content, identification, and modification represents a fundamental assay in biochemistry research. Different analytical techniques and protocols have been specifically designed but have rarely been compared. Here, we test and compare a variety of methodologies and treatments for the quantification of proteins in Amphistegina lessonii, a larger symbiont-bearing benthic foraminiferal species. These analyses specifically include (a) lysis buffer (homemade vs. RIPA), (b) protein assays (Lowry, BCA, and Bradford), (c) ultrasonic bath treatment, and (d) protein staining (silver staining vs. Coomassie blue). On the basis of the comparative outcome, we suggest using the homemade lysis buffer, Lowry or BCA assays, ultrasonic bath treatment, and silver stain to maximize the extraction and characterization of protein for A. lessonii. This protocol might be suitable and extended to other benthic foraminiferal species, including the smaller ones.

Carvacrol effect on topotecan cytotoxicity in various human cancer cells in vitro

Purpose: To investigate the modulatory effect of the natural phytochemical, carvacrol, on Topotecan (TOPO) cytotoxicity and cellular uptake in different cancer cell lines. Methods: The cytotoxicity of the carvacrol/TOPO combination therapy was determined in vitro using crystal violet assay. Coomassie blue and DAPI fluorescent stains were used for cellular morphology and molecular cell death assessments, respectively. Additionally, TOPO cellular uptake after carvacrol/TOPO combination therapy was determined. Results: Treatment of HeLa and HCT116 with carvacrol/TOPO resulted in 7.70- and 5.71-fold reduction in TOPO half maximal inhibitory concentration (IC50), respectively, relative to TOPO single treatment. On the other hand, treatment of MCF-7, HepG2, SKOV3, and A549 cancer cells with carvacrol/TOPO resulted in increasing the IC50 of TOPO by 1.49-, 1.33-, 1.50- and 1.26-fold, respectively, relative to TOPO single treatment. Conclusion: Carvacrol had enhanced TOPO cytotoxicity and cellular uptake in HeLa and HCT116 cancer cells but might cause TOPO resistance in MCF-7, HepG2, SKOV3 and A549 cells.