The Protein Extraction from Longan Pulp (Dimocarpus longan Lour. cv. Daw) for Proteomic Analysis Using One-Dimensional Electrophoresis

Three procedures for protein extraction in longan pulp had been applied to analyze protein pattern and quality of Longan pulp (Dimocarpus longan Lour. cv. Daw) during fruit growth to increase protein expression in proteomic analysis at Maejo university’s farm. There were data points to compare between normal and physiological disorder syndromes during fruit growth (5,10, 15, 20, 25 and 30 weeks, respectively) by one dimensional electrophoresis (1-D gel) technique in reducing condition. The first protein extraction, M1 (95% ethanol) showed obviously 15 protein bands which molecular weights were 14.97, 17.90, 18.30, 21.63, 28.54, 31, 33.96, 35.02, 42, 51.69, 65.69, 71.54, 88.02, 106.86 and 130 kDa, respectively. While M2 extraction (phenol-methanol/ammonium acetate) and M3 extraction (1.5 mM tris-HCl pH 8.0, 5 mM EDTA, 2% SDS) had low protein expression and no sharpness (13 and 12 protein bands, respectively). In different extraction conditions, therefore, M1 was a suitable method because of highest protein bands and obvious protein expression on longan pulp for proteomic analysis. Proteomic analysis of M1 extraction method was used in protein analysis by using LC-MS / MS techniques. It was found that the heat shock protein 83 (81.0 kDa), a family of proteins that was produced by cells in response to exposure on stressful conditions, the elongation factor 1-alpha (49.45 kDa), a selective regulator of translation, and the peroxidase 4 (39.74 kDa), a protein that is involved in the degeneration or aging of cells. These proteins exhibited a darker appearance of the protein bands at 30 weeks. Moreover, a partial glyceraldehyde-3-phosphate dehydrogenase (34.06 kDa), the protein involved in metabolic processes in glucose degradation, was also founded a darker appearance at 25 weeks and low appearance at 30 weeks of abnormal longan. However, higher proteomic techniques should be studied to confirm this biomarker protein in the further.

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Standardization of Sample Preparation for Two-Dimensional Electrophoresis of Cryptosporidium parvum Sporozoites

Bangladesh Journal of Microbiology ◽

10.3329/bjm.v24i2.1255 ◽

1970 ◽

Vol 24 (2) ◽

pp. 119-124

Author(s):

AMAM Zonaed Siddiki

Keyword(s):

Sample Preparation ◽

Proteomic Analysis ◽

Host Location ◽

Protein Extraction ◽

Therapeutic Agents ◽

Automated Image Analysis ◽

Two Dimensional ◽

Two Dimensional Electrophoresis ◽

Dimensional Electrophoresis ◽

Important Parasite

Cryptosporidium is an important parasite of human and animals that belongs to the group Apicomplexa. However, the organism diverges from other Apicomplexa in several important aspects such as its unusual host location, atypical developmental biology, unique metabolism and recently described relict mitochondria. All or some of these unique features may be responsible for the lack of effective therapeutic agents against this parasite. The recent completion of genome sequence projects for C. parvum and C. hominis facilitated postgenomic investigations including proteomic analysis. Sample preparation is the first important step for any proteomic analysis. In this study we have attempted to develop the suitable sample preparation protocol for successful two-dimensional electrophoresis (2-DE) of Cryptosporidium sporozoite proteins prior to mass spectrometry. The 2-DE gels were analysed by automated image analysis software and number of protein spots were used as the indicator for maximum protein extraction from Cryptosporidium sporozoite samples. Keywords: Proteomics, Cryptosporidium, Two-dimensional electrophoresis (2-DE), SolubilizationDOI: http://dx.doi.org/10.3329/bjm.v24i2.1255 Bangladesh J Microbiol, Volume 24, Number 2, December 2007, pp 119-124

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An efficient protein extraction method applied to mangrove plant Kandelia obovata leaves for proteomic analysis

Plant Methods ◽

10.1186/s13007-021-00800-y ◽

2021 ◽

Vol 17 (1) ◽

Author(s):

Jiao Fei ◽

You-Shao Wang ◽

Hao Cheng ◽

Yu-Bin Su

Keyword(s):

Proteomic Analysis ◽

Protein Extraction ◽

Elongation Factor ◽

Extraction Methods ◽

Vital Role ◽

Kandelia Obovata ◽

Protein Patterns ◽

Mangrove Plant ◽

B Method ◽

Flight Mass Spectrometry

Abstract Background Mangroves plants, an important wetland system in the intertidal shores, play a vital role in estuarine ecosystems. However, there is a lack of a very effective method for extracting protein from mangrove plants for proteomic analysis. Here, we evaluated the efficiency of three different protein extraction methods for proteomic analysis of total proteins obtained from mangrove plant Kandelia obovata leaves. Results The protein yield of the phenol-based (Phe-B) method (4.47 mg/g) was significantly higher than the yields of the traditional phenol (Phe) method (2.38 mg/g) and trichloroacetic acid-acetone (TCA-A) method (1.15 mg/g). The Phe-B method produced better two-dimensional electrophoresis (2-DE) protein patterns with high reproducibility regarding the number, abundance and coverage of protein spots. The 2-DE gels showed that 847, 650 and 213 unique protein spots were separated from the total K. obovata leaf proteins extracted by the Phe-B, Phe and TCA-A methods, respectively. Fourteen pairs of protein spots were randomly selected from 2-DE gels of Phe- and Phe-B- extracted proteins for identification by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/TOF-MS) technique, and the results of three pairs were consistent. Further, oxygen evolving enhancer protein and elongation factor Tu could be observed in the 2-DE gels of Phe and Phe-B methods, but could only be detected in the results of the Phe-B methods, showing that Phe-B method might be the optimized choice for proteomic analysis. Conclusion Our data provides an improved Phe-B method for protein extraction of K. obovata and other mangrove plant tissues which is rich in polysaccharides and polyphenols. This study might be expected to be used for proteomic analysis in other recalcitrant plants.

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Membrane and Secretory Protein Extraction of Mycobacterium Tuberculosis and Mycobacterium Bovis Using One Dimensional Electrophoresis (SDS-PAGE)

Journal of Clinical Microbiology and Biochemical Technology ◽

10.17352/jcmbt.000025 ◽

2017 ◽

pp. 040-044

Author(s):

AH Tasbiti

Keyword(s):

Mycobacterium Tuberculosis ◽

Mycobacterium Bovis ◽

Protein Extraction ◽

Secretory Protein ◽

One Dimensional ◽

Sds Page ◽

Dimensional Electrophoresis

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Faculty Opinions recommendation of Proteomic analysis of acetaminophen-induced changes in mitochondrial protein expression using spectral counting.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.8632956.9136054 ◽

2011 ◽

Author(s):

Philip Burcham

Keyword(s):

Protein Expression ◽

Proteomic Analysis ◽

Mitochondrial Protein ◽

Spectral Counting ◽

Induced Changes

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Mass Spectrometry-based Label-free Quantitative Proteomic Analysis of CCl4-induced Acute Liver Injury in Mice Intervened by Total Glycosides from Ligustri Lucidi Fructus

Current Proteomics ◽

10.2174/1570164617999200728192812 ◽

2020 ◽

Vol 17 ◽

Author(s):

Qian Lu ◽

Hai-Zhu Xing ◽

Nian-Yun Yang

Keyword(s):

Insulin Resistance ◽

Liver Injury ◽

Protein Expression ◽

Signaling Pathway ◽

Proteomic Analysis ◽

Acute Liver Injury ◽

Liver Cells ◽

Differentially Expressed ◽

Liver Protein ◽

Differentially Expressed Proteins

Background: CCl4 acute liver injury (ALI) is a classical model for experimental research. However, there are few reports involved in the fundamental research of CCl4-induced ALI Ligustri Lucidi Fructus (LLF) are and its prescription have been used to treat hepatitis illness clinically. LLF and its active ingredients displayed anti-hepatitis effects, but the mechanism of function has not been fully clarified Objective: To investigate the proteomic analysis of CCl4-induced ALI, and examine the effects of active total glycosides (TG) from LLF on ALI of mice4, including histopathological survey and proteomic changes of liver tissues, and delineate the possible underlying mechanism. Methods: CCl4 was used to produce ALI mice model. The model mice were intragastrically administrated with TG and the liver his-topathological changes of mice were examined. At the end of test, mice liver samples were collected, after protein denaturation, re-duction, desalination and enzymatic hydrolysis, identification was carried out by nano LC-ESI-OrbiTrap MS/MS technology. The data was processed by Maxquant software. The differentially-expressed proteins were screened and identified, and their biological information was also analyzed based on GO and KEGG analysis. Key protein expression was validated by Western blot analysis Results: A total of 705 differentially-expressed proteins were identified during the normal, model and administration group. 9 signifi-cant differential proteins were focused based on analysis. Liver protein expression changes of CCl4-induced ALI mice were mainly involved in several important signal channels, namely FoxO signaling pathway, autophagy-animal, insulin signaling pathway. TG has anti-liver damnification effect in ALI mice, the mechanism of which is related to FoxO1 and autophagy pathways Conclusion: CCl4 inhibited expression of insulin-Like growth factor 1 (Igf1) and 3-phosphoinositide-dependent protein kinase 1 (Pdpk1) in liver cells and induced insulin resistance, thus interfered with mitochondrial autophagy and regeneration of liver cells and the metabolism of glucose and lipid, and caused hepatic necrosis in mice. TG resisted liver injury in mice. TG adjusted the expression level of key proteins Igf1 and Pdpk1 after liver injury and improved insulin resistance, thus promoted autophagy and resisted the liver damage

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Proteomic analysis on insulin signaling in human hematopoietic cells: identification of CLIC1 and SRp20 as novel downstream effectors of insulin

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.00512.2004 ◽

2005 ◽

Vol 289 (3) ◽

pp. E419-E428 ◽

Cited By ~ 25

Author(s):

Kumiko Saeki ◽

Etsuko Yasugi ◽

Emiko Okuma ◽

Samuel N. Breit ◽

Megumi Nakamura ◽

...

Keyword(s):

Western Blotting ◽

Proteomic Analysis ◽

Intracellular Signaling ◽

Hematopoietic Cells ◽

Chloride Ion ◽

Qualitative Change ◽

Insulin Stimulation ◽

One Dimensional ◽

Early Protein ◽

Downstream Effectors

Insulin/IGF-I-dependent signals play important roles for the regulation of proliferation, differentiation, metabolism, and autophagy in various cells, including hematopoietic cells. Although the early protein kinase activation cascade has been intensively studied, the whole picture of intracellular signaling events has not yet been clarified. To identify novel downstream effectors of insulin-dependent signals in relatively early phases, we performed high-resolution two-dimensional electrophoresis (2-DE)-based proteomic analysis using human hematopoietic cells 1 h after insulin stimulation. We identified SRp20, a splicing factor, and CLIC1, an intracellular chloride ion channel, as novel downstream effectors besides previously reported effectors of Rho-guanine nucleotide dissociation inhibitor 2 and glutathione S-transferase-pi. Reduction in SRp20 was confirmed by one-dimensional Western blotting. Moreover, MG-132, a proteasome inhibitor, prevented this reduction. By contrast, upregulation of CLIC1 was not observed in one-dimensional Western blotting, unlike the 2-DE results. As hydrophilic proteins were predominantly recovered in 2-DE, the discrepancy between the 1-DE and 2-DE results may indicate a certain qualitative change of the protein. Indeed, the nuclear localization pattern of CLIC1 was remarkably changed by insulin stimulation. Thus insulin induces the proteasome-dependent degradation of SRp20 as well as the subnuclear relocalization of CLIC1.

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