Coomassie Brilliant Blue Staining Research Articles

Proteomic analysis of heat shock proteins in maize (Zea mays L.)

BackgroundMaize is one of the important cereal food crops in the world. High temperature stress causes adverse influence on plant growth. When plants are exposed to high temperatures, they produce heat shock proteins (HSPs), which may impart a generalized role in tolerance to heat stress. Proteome analysis was performed in plant to assess the changes in protein types and their expression levels under abiotic stress. The purpose of the study is to explore which proteins are involved in the response of the maize plant to heat shock treatment.ResultsWe investigated the responses of abundant proteins of maize leaves, in an Egyptian inbred line of maize “K1”, upon heat stress through two-dimensional electrophoresis (2-DE) on samples of maize leaf proteome. 2-DE technique was used to recognize heat-responsive protein spots using Coomassie Brilliant Blue (CBB) and silver staining. In 2-D analysis of proteins from plants treated at 45 °C for 2 h, the results manifested 59 protein spots (4.3%) which were reproducibly detected as new spots where did not present in the control. In 2D for treated plants for 4 h, 104 protein spots (7.7%) were expressed only under heat stress. Quantification of spot intensities derived from heat treatment showed that twenty protein spots revealed clear differences between the control and the two heat treatments. Nine spots appeared with more intensity after heat treatments than the control, while four spots appeared only after heat treatments. Five spots were clearly induced after heat treatment either at 2 h or 4 h and were chosen for more analysis by LC-MSMS. They were identified as ATPase beta subunit, HSP26, HSP16.9, and unknown HSP/Chaperonin.ConclusionThe results revealed that the expressive level of the four heat shock proteins that were detected in this study plays important roles to avoid heat stress in maize plants.

Targeted Early Detection of Citrus Huanglongbing Causal Agent 'Candidatus Liberibacter asiaticus' Before Symptom Expression.

Citrus Huanglongbing (HLB) is the most severe disease of citrus plants caused by 'Candidatus Liberibacter asiaticus' and transmitted by the insect vector Asian citrus psyllid (ACP). No effective curative measure is available against HLB. For citrus production areas without HLB or with low HLB disease incidence, removal of 'Ca. L. asiaticus' inoculum is critical to prevent HLB spread. Such a strategy requires robust early diagnosis of HLB for inoculum removal to prevent ACP acquisition and transmission of 'Ca. L. asiaticus'. However, early diagnosis of HLB is challenging, because the citrus trees remain asymptomatic for several months to years after 'Ca. L. asiaticus' transmission by ACP. In this study, we report a new method for targeted early detection of 'Ca. L. asiaticus' in cultivar Valencia sweet orange (Citrus sinensis) before HLB symptom expression. We take advantage of the fact that 'Ca. L. asiaticus' remains around the ACP feeding site immediately after transmission into the young flush and before flush maturation. ACPs secrete salivary sheaths at their feeding sites, which can be visualized using Coomassie brilliant blue staining owing to the presence of salivary sheaths secreted by ACP. Epifluorescence and confocal microscopy indicate the presence of salivary sheaths beneath the blue spots on ACP-fed leaves. Quantitative real-time polymerase chain reaction (PCR) and conventional PCR assays are able to detect 'Ca. L. asiaticus' in the ACP feeding surrounding areas as early as 2 to 20 days after ACP feeding. This finding lays a foundation to develop much-needed tools for early diagnosis of HLB before symptom expression, thus assisting 'Ca. L. asiaticus' inoculum removal and preventing HLB from spreading.

Enolase1 Alleviates Cerebral Ischemia-Induced Neuronal Injury via Its Enzymatic Product Phosphoenolpyruvate.

Stroke is a leading cause of disability and the second leading cause of death among adults worldwide, while the mechanisms underlying neuronal death and dysfunction remain poorly understood. Here, we investigated the differential proteomic profiles of mouse brain homogenate with 3 h of middle cerebral artery occlusion (MCAO) ischemia, or sham, using Coomassie Brilliant Blue staining, followed by mass spectrometry. We identified enolase1 (ENO1), a key glycolytic enzyme, as a potential mediator of neuronal injury in MCAO ischemic model. Reverse transcription polymerase chain reaction and western blotting data showed that ENO1 was ubiquitously expressed in various tissues, distinct regions of brain, and different postnatal age. Immunohistochemical analysis revealed that ENO1 is localized in neuronal cytoplasm and dendrites. Interestingly, the expression level of ENO1 was significantly increased in the early stage, but dramatically decreased in the late stage, of cerebral ischemia in vivo. This dynamic change was consistent with our finding in cultured hippocampal neurons treated with oxygen/glucose deprivation (OGD) in vitro. Importantly, ENO1 overexpression in cultured neurons alleviated dendritic and spinal loss caused by OGD treatment. Furthermore, the enzymatic product of ENO1, phosphoenolpyruvate (PEP), was also synchronously changed along with the dynamic ENO1 level. The neuronal injury caused by OGD treatment in vitro or ischemia in vivo was mitigated by the application of PEP. Taken together, our data revealed that ENO1 plays a novel and protective role in cerebral ischemia-induced neuronal injury, highlighting a potential of ENO1 as a therapeutic target of neuronal protection from cerebral ischemia.

Therapeutic effects of baicalin on lupus nephritis in a lupus-prone mouse model and its regulatory role in follicular helper T cell differentiation

Objective To evaluate the therapeutic effect of baicalin on lupus nephritis in a lupus-prone mouse model, and to investigate its regulatory role in the differentiation of follicular helper T (Tfh) cells. Methods Eight 12-week-old female MRL/lpr lupus-prone mice were randomly and equally divided into two groups by a random number table i.e., baicalin group and control group intraperitoneally injected with 200 mg/kg baicalin sodium and chloride physiological solution, respectively, once every day for 4 weeks. After the end of treatment, Coomassie brilliant blue staining was performed to detect the level of 24-hour urine protein. Then, the mice were sacrificed, and the spleens were resected and weighed. Mononuclear cells were isolated from these spleens, and flow cytometry was conducted to determine the proportion of Tfh cells. Additionally, the kidneys were resected and subjected to hematoxylin and eosin (HE) staining for the evaluation of kidney impairment. Moreover, some other mononuclear cells were isolated from the spleens of the lupus-prone mice in the control group, and magnetic activated cell sorting (MACS) was performed to isolate naive CD4+ T cells, which were divided into 3 groups: blank control group receiving no treatment, induction group treated with 10 μg/L anti-interleukin (IL) -21 and anti-IL-6 antibodies and 3 μg/L anti-CD3 and anti-CD28 antibodies for 5 days, and intervention group additionally treated with 40 μmol/L baicalin for 5 days besides the above treatment. Then, 50 μg/L phorbol ester, 750 μg/L ionomycin and 20 mg/L brefeldin A were used to stimulate some cultured naive CD4+ T cells in the above groups. Flow cytometry was conducted to determine the proportion of CD4+CXCR5+PD-1+ cells and CD4+IL-21+ cells. Statistical analysis was carried out with SPSS20.0 software by using one-way analysis of variance (ANOVA) and student t test for the comparison of quantitative data between groups. Results The baicalin treatment could effectively improve the kidney impairment in the lupus-prone mice. Compared with the control group, the baicalin group showed significantly decreased 24-hour urine protein level ([1 416 ± 171] vs. [2 623 ± 278] μg/24 h, P = 0.022) , and significantly decreased proportion of Tfh cells in the spleen (12.6% ± 2.3% vs. 40.2% ± 1.1%, P = 0.005) . In vitro baicalin could further inhibit the differentiation of Tfh cells. Compared with the induction group, the intervention group showed significantly decreased proportion of CD4+CXCR5+PD-1+ Tfh cells (13.3% ± 0.8% vs. 17.6% ± 0.9%, P = 0.04) and CD4+IL-21+ cells (1.0% ± 0.4% vs. 2.7% ± 0.2%, P < 0.01) . Conclusion Baicalin can effectively ameliorate lupus nephritis, which may be associated with the inhibition of Tfh cell differentiation. Key words: Lupus erythematosus, systemic; Lupus nephritis; Baicalin; Disease models, animal; Follicular helper T cells; Interleukin-21

Protein Biomarkers from Physiological Disorder Syndrome in Leave, Peel and Seed of Longan on Fruit Growth Using One-Dimension Electrophoresis Technique Followed by Protein Identification with LC-MS/MS

Proteomic investigation of leaves, peels and seeds on longan (Dimocarpus longan Lour. cv. Daw) at Maejo University’s farm in Chiang Mai province, Thailand. There were comparisons between normal and physiological disorder syndromes in longan on fruit growth (5, 10, 15, 20, 25 and 30 weeks fruition, respectively) by one dimensional electrophoresis (1-D gel) technique at a concentration of 12.5% gel in reducing condition with Coomassie brilliant blue R-250 staining coupling on liquid chromatography tandem mass spectrometry (LC-MS/MS). The research showed that vegetative storage protein (25.2 KDa), a storage protein in plants, was expressed in leaves, peels and seeds on before and after physiological disorder syndrome occurring. A protein involved on photosynthesis, the ribulose-1,5-bisphosphate carboxylase/oxygenase (48.3 KDa), was represented only on leaves at 30 weeks after fruition but not in peels and seeds of longan. Two proteins, the BnaC01g20210 (35.8 KDa) and predicted protein (40.11 KDa), which could not be identified the type and function of the metabolism, were decreased in diseased longan. These proteins may be important protein in part of the recovery process of abnormal longan. Therefore, advanced technique will be used for further proteomic studies.

Study on Dormancy Mechanism and Breaking Dormancy Method of &amp;lt;i&amp;gt;Viburnum sargentii&amp;lt;/i&amp;gt; Seeds

Under untreated conditions, the germination rate of Viburnum sargentii seeds is very low. By exploring the relationship between the dynamic changes of physicochemical indicators and endogenous hormones and seed germination during the dormancy and germination of Viburnum sargentii seeds, the mechanism of seed germination of Viburnum sargentii was determined, which provided a theoretical basis for its extensive promotion and development. The contents of soluble sugar and soluble starch were determined by anthrone colorimetry. The soluble protein was determined by Coomassie brilliant blue G-250 staining. The activity of polyphenol oxidase (PPO) was determined by NBT reduction method. The peroxidase (POD) was determined by guaiacol method. And the endogenous hormones ZA, IAA, ABA, GA3 were determined by liquid chromatography. Results show: 1) Under natural conditions, the seeds of Viburnum sargentii are difficult to germinate. 2) Under sand storage for 8 months, the germination rate of untreated seeds was 33%. Puncture and peeling treatment could significantly increase the germination rate, and the germination rate of seeds treated with puncture was 92%, and that treated with peel was 98%. 3) Seed germination was accompanied by the decrease in macromolecular substances such as soluble sugar, soluble starch, and soluble protein. 4) The dynamic changes of hormones during seed germination conform to the hypothesis of “three factors”. 5) The treatment of puncture and peeling increased the content of endogenous hormones promoting germination, decreased the endogenous hormones inhibiting seed germination, and increased the ratio of (IAA + GA + ZR)/ABA or GA/ABA. The seeds of Viburnum sargentii have obvious dormancy characteristics. Under the condition of sand storage, both pricking and peeling treatment can effectively promote the process of breaking dormancy, and the effect of peeling treatment is better.

Transgelin as a potential target in the reversibility of pulmonary arterial hypertension secondary to congenital heart disease.

BackgroundThe reversibility of pulmonary arterial hypertension (PAH) in congenital heart disease (CHD) is of great importance for the operability of CHD. Proteomics analysis found that transgelin was significantly up‐regulated in the lung tissue of CHD‐PAH patients, especially in the irreversible group. However, how exactly it participated in CHD‐PAH development is unknown.MethodsImmunohistochemical staining and Western blot were performed for further qualitative and quantitative analysis of transgelin in the lung tissues of CHD‐PAH patients. The mechanism of transgelin in CHD‐PAH development was explored in vitro. Primary human pulmonary arterial smooth muscle cells (hPASMCs) were cultured and infected with TAGLN siRNA or TAGLN lentiviral vector. Cell morphologic change (Coomassie Brilliant Blue staining), proliferation (cell count and EdU assay), apoptosis (terminal deoxyribonucleotidyl transferase mediated dUTP nick end labeling assay and Annexin‐V flow cytometry) and migration (transwell) were evaluated following the cell treatment. The mRNA and protein expression levels were detected in real‐time PCR and Western blot.ResultsIn line with the proteomic findings, transgelin was obviously expressed in PASMC of the middle pulmonary arterioles, especially in the irreversible PAH group. Also, transgelin expression showed positive relation with pathological grading. Experiment in vitro demonstrated that transgelin overexpression promoted PASMC proliferation and migration, strengthened cytoskeleton and was accompanied by increased expression of synthetic phenotype markers (osteopontin, proliferating cell nuclear antigen) and anti‐apoptotic protein (bcl‐2). On the other hand, suppression of transgelin expression activated PASMC apoptosis, reducing cell proliferation and migration.ConclusionsTransgelin may be a potential target in the development of irreversible CHD‐PAH through inducing PASMC phenotype change, proliferation, migration and reducing cell apoptosis.

User and environment friendly direct agglutination test for the sero-diagnosis of visceral leishmaniasis: exclusion of formaldehyde and β-mercaptoethanol in test execution.

Based on world-wide evaluation, the direct agglutination test (DAT) is now generally acknowledged as one of the leading diagnostics for visceral leishmaniasis (VL). To enhance more routine and mass application, but simultaneously ensure safety to both user and environment, further improvements need to be introduced. In the current format, a two-sixfold titre decrease was observed due to using formaldehyde as an antigen preservative in DAT. Successful formaldehyde preservative exclusion was achieved by increasing its concentration to 3 % (wt/vol) for conserving promastigote status after β-mercaptoethanol (β-ME) treatment and repeating exposure of the parasite to the fixative after Coomassie Brilliant Blue staining. Microbial contamination was not observed in any of the antigen aliquots preserved in 0.05 % (wt/vol) sodium dichloroisocyanurate (chlorine) instead of formaldehyde for 6 months or longer. By excluding formaldehyde, restoring the normal antibody level, prior to treatment of sera with β-ME only minimally influenced the test outcome. A comparable successful reduction in non-specific agglutination, as with β-ME, was achieved by incorporating urea (0.3 % wt/vol) in the improved DAT procedure (P=0.646; T=23.0). As with the current procedure, the improved equivalent (formaldehyde and β-ME free) showed good reliability for VL detection (VL - Fr=52.39, W=0.70, P<0.001; and non-VL - Fr=65.97, W=0.83, P<0.001). A much lower cut-off (titre 1 : 400 versus 1 : 3200) for VL diagnosis can be adopted if urea is integrated in the improved procedure. By introducing the modifications mentioned, we think we have succeeded to a reasonable degree in increasing the DAT potential for VL control.

Worm wounding increases levels of pollen-related food allergens in soybean (Glycine max)

The levels of food allergens in worm-wounded or non-wounded green soybeans (edamame) and mature soybeans were investigated by immunoblotting and enzyme-linked immunosorbent assay (ELISA), using allergen-specific antibodies. Non-wounded and worm-wounded soybeans showed similar total protein profiles after Coomassie brilliant blue staining, but some protein bands were observed to have been changed by worm wounding. Immunoblotting with specific antibodies for major soybean allergens (Gly m 5, Gly m 6, Gly m Bd 30K, and Kunitz soybean trypsin inhibitor) revealed that protein band profiles and intensities were not significantly changed by worm wounding. In contrast, levels of the pollen-related soybean allergens Gly m 4 and Gly m 3 were strongly increased by worm wounding in both green and mature soybeans, as detected by immunoblotting and ELISA. These results suggested that the pollen-related food allergen risk (i.e., oral allergy syndrome; OAS) from soybeans might be enhanced by worm wounding of soybeans.

Coomassie does it (better): A Robin Hood approach to total protein quantification

Quantitative comparative proteomics require accurate and reproducible assessments of total protein concentration. We report a straightforward, cost-effective adaptation of an established commercial method for total protein quantification (EZQ™), utilising non-proprietary materials and colloidal Coomassie Brilliant Blue (cCBB) staining to achieve greater reproducibility, equal sensitivity, and optimal linearity of signal within a practical concentration range for proteins in common solubilisation buffers (i.e. for isoelectric focussing and/or SDS-PAGE). This method provided more accurate and precise determinations of total protein concentration in human serum prepared for two-dimensional gel electrophoresis, indicating it may be better suited as the lead-in to most quantitative proteomic analyses.

Analysis of new charge-neutral DNA/RNA analogues phosphoryl guanidine oligonucleotides (PGO) by gel electrophoresis

Analysis and isolation of new charge-neutral phosphoryl guanidine oligonucleotides (PGO) by vertical slab electrophoresis were tested at different pH values (3–11) or in the presence of SDS as a micelle-forming agent. The most convenient way to analyze and purify phosphoryl guanidine oligonucleotides was by denaturing PAGE (8 M urea) at pH 3. The mobility of PGO is dependent on their A + C content. To analyze PGO containing only G, T or U, denaturing PAGE at pH 11 can be used, although the conditions need to be optimized. Bands were visualized by UV shadowing or Coomassie Brilliant Blue staining.

High Sensitivity Top‐down Proteomics: Coomassie for In‐gel Proteoform Detection Rivals MS‐based Peptide Detection

Several thousands of gene and splice variants, as well as post‐translational modifications, deepen the immense complexity of the human proteome. As these protein species or proteoforms define biological mechanisms in health and disease their direct analysis is critical, necessitating top‐down assessments in which the complete amino acid sequence and all modifications remain intact. Two‐dimensional gel electrophoresis (2DE) coupled with downstream mass spectrometry (MS) remains the most thorough and practical approach to Top‐down Discovery Proteomics, with resolving power to routinely analyze complex mixtures of intact proteoforms [1]. Increasing the quality and quantity of 2DE‐derived data largely relies on developing, optimising, and rigorously characterising methods for in‐gel total protein detection, providing quantitative, sensitive detection over a wide dynamic range while remaining practical [2]. Modified colloidal Coomassie Brilliant Blue (cCBB) staining meets these criteria [3, 4]; cCBB provides (sub)femtomole detection sensitivity for SDS‐PAGE and 2DE‐resolved intact proteoforms using either near‐infrared fluorescence detection (nIRFD) or densitometry [5]. This thus rivals the detection sensitivity of peptides by MS in ‘bottom‐up’ (i.e. shotgun) approaches to proteomics, which appear to provide larger protein catalogues but at the expense of critical data concerning proteoforms. Our ongoing work addresses a number of experimental variables which impact detection sensitivity in standard 1 mm‐thick polyacrylamide gels, providing a more realistic baseline measure of cCBB detection sensitivity and enhancing the overall practicality of 2DE for comprehensive proteome analysis. Here we showcase our most recent findings, including preliminary findings of further improvements to cCBB detection sensitivity when using ultra‐thin polyacrylamide gels. The novel ultra‐thin method utilised has been developed from minimally modified commercial equipment for vertical mini‐format gel electrophoresis and is thus accessible, affordable, and simple to implement in laboratories equipped for standard SDSPAGE and 2DE. The ultra‐thin gels do not require backing supports for handling, and potentially are compatible with commercial immobilised pH gradient strips routinely used for first dimension isoelectric focussing, further adding to the value and practicality of the method. Ultra‐thin 2DE may thus enable further mining of 2DE‐resolved proteomes, including the application of high resolution gel‐based proteomics in situations of limited sample quantity (e.g. biopsies, and perhaps single cells).Support or Funding InformationNN and PSA received scholarship support from the WSU Molecular Medicine Research Group and the WSU School of Medicine, respectively. JRC notes the support of an anonymous private family foundation.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Paper Adsorbents Remove Coomassie Blue from Gel Destain and Used Gel Stain in anEnvironment-Friendly Manner.

Coomassie Brilliant Blue (CBB), used to stain protein gels, is known to be toxic. Therefore, laboratories do not discard used CBB into the sink owing to the possibility of it contaminating drinking water supplies. We tested the ability of various paper adsorbents to adsorb CBB released from gels during destaining. The efficiency was as follows-Kimwipes>Teri towels>multifold towels>Whatman numbers 1 and 3 filter papers. Addition of three Kimwipes during destaining helped adsorb the dye released from a CBB-stained mini-gel. Stain removal with Kimwipes helps reduce destain use, organic waste accumulation, enable recycling of nonradioactive destaining solution and is 7.5-fold cheaper than an available method for CBB disposal. Next, we used Kimwipes to deplete the dye from a used CBB staining solution awaiting proper disposal by our Institutional Safety Office. Seventy five Kimwipes successfully helped remove the dye from a 0.05% CBB staining solution in 5-10 min. The blue Kimwipes did not release the CBB stain even when squeezed dry after incubation in various salts, water, or acid solutions for 5weeks. The CBB removed thus can be simply disposed of as solid waste and will not leach out from solid landfills. Kimwipes, thus, enables CBB disposal in an environmentally friendly manner and allows for recycling of destaining solution.

Coomassie Brilliant Blue Staining of Polyacrylamide Gels.

In the past a series of staining procedures for proteins were published. Still, the most commonly used staining dye for proteins is Coomassie Brilliant Blue. The major reason is that Coomassie Brilliant Blue staining is simple, fast, and sensitive. As Coomassie Brilliant Blue is almost insoluble in water a series of procedures including colloidal aqueous procedures has been described.

An efficient 2-D gel electrophoresis protocol suitable for seed proteome of rice

The aim of this article was to provide the efficient protocol for two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) suitable for the seed proteome of rice. Sample preparation is a crucial step in 2D-PAGE because it directly affects the quality of protein profiles. We investigated the two different methods to extract the proteins viz., direct lysis buffer method and trichloroacetic acid/acetone (TCA/acetone) method. The effectiveness was evaluated by the protein profing quality, number of protein spots, consistency and distribution of protein spots over the gels. The protein extracted by TCA/acetone method had the highest number of protein spots and the good resolution of the gels. Two different IEF focusing were tested in which, the protein run at 50000 Volt hours showed the clear protein pattern and more number of proteins than 35000 Volt hours. Compared to the Coomassie brilliant blue (CBB) G-250 staining, silver nitrate staining produced the more number of proteins spots. The protocol using TCA/Acetone extraction with 50000 Volt-hour IEF conditions followed by the silver staining resulted in the best extraction efficiency, greater protein separation and less horizontal streaking in 2DE gels. This works could principally provide the useful information and technical support for the rice seed proteomic research.

A One-Step Staining Protocol for In-Gel Fluorescent Visualization of Proteins.

Native polyacrylamide gel electrophoresis (PAGE) and sodium dodecyl sulfate (SDS)-PAGE are among the most frequently applied techniques in protein analysis. Here we describe a fast one-step method for fluorescent visualization of proteins. Following PAGE, gels are soaked in solution of potassium ferricyanide (100mM) in 1 M NaOH, and are kept in the dark for 30 min. Gels are then transferred to water and scanned. The sensitivity of the method is comparable with standard Coomassie Brilliant Blue staining.

Heterologous expression, purification and function of the extracellular domain of human RANK

BackgroundReceptor activator of NF-κB ligand (RANKL)/RANK signaling essentially functions within the skeletal system, particularly participating in osteoclastogenesis and bone resorption. In addition, this signaling pathway has also been shown to influence tumor progression as well as the development and function of the immune system. Therefore, blocking the interaction between RANKL and RANK is a new therapeutic approach to prevent bone-related diseases and cancer.ResultsThe coding sequence encoding the extracellular domain of human RANK (RANK-N) was codon optimized for Pichia pastoris and cloned into the pPIC9K vector, and the recombinant plasmid was then transformed into P. pastoris. The expression of RANK-N protein was confirmed using SDS-PAGE with Coomassie Brilliant Blue stain and western blotting. Recombinant RANK-N protein was purified by a multistep process including ultrafiltration (UF), Sephadex G-50 size-exclusion chromatography and Q-Sepharose Fast Flow ion exchange chromatography, which resulted in a purity >95%. We found that the RANK-N protein can block RANKL-RANK signaling both in vitro and in vivo. Furthermore, using a patient-derived xenograft of human colon cancer, we found that the recombinant RANK-N protein can inhibit the growth of colorectal cancer.ConclusionsThe results show that a simple system to express and purify functional RANK-N protein has been developed. This work has thus laid a foundation for further research and clinical applications of RANK-N protein in treating bone-related diseases or even colorectal cancer.

Coomassie staining provides routine (sub)femtomole in-gel detection of intact proteoforms: Expanding opportunities for genuine Top-down Proteomics.

Modified colloidal Coomassie Brilliant Blue (cCBB) staining utilising a novel destain protocol and near-infrared fluorescence detection (nIRFD) rivals the in-gel protein detection sensitivity (DS) of SYPRO Ruby. However, established DS estimates are likely inaccurate in terms of 2DE-resolved proteoform 'spots' since DS is routinely measured from comparatively diffuse protein 'bands' following wide-well 1DE. Here, cCBB DS for 2DE-based proteomics was more accurately determined using narrow-well 1DE. As precise estimates of protein standard monomer concentrations are essential for accurate quantitation, coupling UV absorbance with gel-based purity assessments is described. Further, as cCBB is compatible with both nIRFD and densitometry, the impacts of imaging method (and image resolution) on DS were assessed. Narrow-well 1DE enabled more accurate quantitation of cCBB DS for 2DE, achieving (sub)femtomole DS with either nIRFD or densitometry. While densitometry offers comparative simplicity and affordability, nIRFD has the unique potential for enhanced DS with Deep Imaging. Higher-resolution nIRFD also improved analysis of a 2DE-resolved proteome, surpassing the DS of standard nIRFD and densitometry, with nIRFD Deep Imaging further maximising proteome coverage. cCBB DS for intact proteins rivals that of mass spectrometry (MS) for peptides in complex mixtures, reaffirming that 2DE-MS currently provides the most routine, broadly applicable, robust, and information-rich Top-down approach to Discovery Proteomics.

Extraction of flocculants from a strain of Bacillus thuringiensis and analysis of their properties

In a preliminary screening study, our laboratory isolated from the biofloc in aquaculture waters a strain of Bacillus thuringiensis, which produced highly efficient bio-flocculants. In the present study, we extracted the crude flocculants from this strain and analyzed their properties. Distribution analysis indicated that the flocculants were mainly distributed in the supernatant of the fermentation liquid. The flocculants were extracted using an ethanol extraction method, and the chemical compositions and morphology of the crude flocculants were analyzed using the Molish reaction, Fehling reaction, ninhydrin reaction, biuret reaction, phenol-sulfuric acid assay, Coomassie brilliant blue staining, ultraviolet scanning, infrared scanning and scanning electron microscopy. The carbohydrate composition of the polysaccharides in the flocculants was analyzed with thin layer chromatography. The results indicated that flocculants were solid substances with an ivory white color and their texture was loose and soft. Visualization under scanning electron microscopy revealed that their ultra-morphology consisted of small, long and fiber-like shapes. Chemical and physical analyses indicated that polysaccharides accounted for 34.5% of the components in the crude flocculants. The monosaccharides present in crude flocculants included mainly glucose, galactose and mannitose.

Phenoloxidase activity in humoral plasma, hemocyanin and hemocyanin separated proteins of the giant freshwater prawn Macrobrachium rosenbergii

Hemocyanin is a copper containing protein and its role in the immune function of phenoloxidase (PO) activity was investigated in the giant freshwater prawn Macrobrachium rosenbergii. Hemocyanin, sedimented by ultracentrifugation from the plasma appeared on polyacrylamide gel electrophoresis (PAGE 7%) on Coomassie Brilliant Blue and bathocuproine sulfonic acid stain as four copper containing proteins of molecular masses 50, 60, 114 and 325kDa. Accordingly, on diethylaminoethyl-cellulose anion exchange column hemocyanin separated into four proteins designated as MrHc1, MrHc2, MrHc3 and MrHc4 with electrophoretically (PAGE) determined molecular masses of 60, 114, 50 and 325kDa respectively. The reduction of proteins in sodium dodecyl sulphate (SDS)-PAGE revealed that MrHc1 and 3 were monomeric for 60and 50kDa respectively, MrHc2 dimeric of 56 and 58kDa subunits and MrHc4 appeared with three subunits of 74, 76 and 78kDa. The PO activity was determined in plasma, hemocyanin and the four separated hemocyanin proteins in vitro using L-3,4-dihydroxyphenylalanine (L-DOPA) at pH7.5, 25°C and appeared elicited by exogenous activators such as trypsin, SDS, cell wall components of bacteria and polysaccharide laminarin. This study clearly demonstrated hemocyanin as the major copper containing protein in the plasma of M. rosenbergii with potent PO activity.