Extraction of proteins from pine needles for proteomic analysis has long been a challenge for scientists. We compared three different protein extraction methods including sucrose, Tris-HCl and trichloroacetic acid （...Extraction of proteins from pine needles for proteomic analysis has long been a challenge for scientists. We compared three different protein extraction methods including sucrose, Tris-HCl and trichloroacetic acid （TCA）/acetone （TCA method） to determine their efficiency in separating pine needle proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis （SDS-PAGE） and two-dimensional PAGE （2D-PAGE）. Proteins were then separated by SDS-PAGE. Among three methods the method using sucrose extraction buffer showed the highest efficiency and highest quality in separating proteins. In addition, clearer and more stable strips were detected by SDS-PAGE using sucrose extraction buffer. When the proteins extracted using sucrose extraction buffer were separated by 2D-PAGE, more than 300 protein spots, with isoelectric points （PI） ranging from 4.0 to 7,0 and molecular weights （MW） from 6.5 to 97.4 kD, were observed. This confirmed that the method with sucrose extraction buffer was an efficient and reliable method for extracting proteins from pine needles.展开更多
Good quality deoxyribonucleic acid （DNA） is the pre-requisite for its downstream applications. The presence of high concentrations of polysaccharides, polyphenols, proteins, and other secondary metabolites in mango ...Good quality deoxyribonucleic acid （DNA） is the pre-requisite for its downstream applications. The presence of high concentrations of polysaccharides, polyphenols, proteins, and other secondary metabolites in mango leaves poses problem in getting good quality DNA fit for polymerase chain reaction （PCR） applications. The problem is exacerbated when DNA is extracted from mature mango leaves. A reliable and modified protocol based on the cetyl trimethylammonium bromide （CTAB） method for DNA extraction from mature mango leaves is described here. High concentrations of inert salt were used to remove polysaccharides; Polyvinylpyrrolidone （PVP） and 13-mercaptoethanol were employed to man＇age phenolic compounds. Extended chloroform-isoamyl alcohol treatment followed by RNase treatment yielded 950-1050 pg of good quality DNA, free of protein and RNA. The problems of DNA degradation,contamination, and low yield due to irreversible binding of phenolic compounds and coprecipitation of polysaccharides with DNA were avoided by this method, The DNA isolated by the modified method showed good PCR amplification using simple sequence repeat （SSR） primers, This modified protocol can also be used to extract DNA from other woody plants having similar problems.展开更多
Bioavailability is a critical factor for assessing the environmental risk of organic pollutants in soil. In this study, extractions with 3 different solvents, including 2 aqueous solutions, calcium chloride(CaCl_2) an...Bioavailability is a critical factor for assessing the environmental risk of organic pollutants in soil. In this study, extractions with 3 different solvents, including 2 aqueous solutions, calcium chloride(CaCl_2) and a phosphate buffer solution(PBS), and a mixture of aqueous solution and organic solvent, a PBS-methanol(8:2,volume/volume) mixture(PBS-M), were performed to assess the bioavailability of chlorimuron-ethyl in soil in comparison to a battery of toxicity tests in wheat seedlings. The results indicated that the peroxidase(POD) activity in wheat leaves after 7 d of exposure was one of the sensitive biomarkers of chlorimuron-ethyl in soil.The extractability of chlorimuron-ethyl by all the 3 solvents decreased with exposure time, and the rate of decrease of the PBS-M extraction between 1 and 7 d of exposure was substantially higher than those of the aqueous solution extractions. Chlorimuron-ethyl gradually changed from a water-soluble form into a soil organic matter(SOM)-bound form in the soil. The PBS extraction correlated best with the POD activity in the leaves after 7 d of exposure.展开更多
Propolis is a resinous natural product,produced by bees(Apis mellifera),from vegetable parts and plant secretions.Propolis’samples A,B,C and D were extracted with phosphate buffer saline(PBS)or with 70%EtOH at pH val...Propolis is a resinous natural product,produced by bees(Apis mellifera),from vegetable parts and plant secretions.Propolis’samples A,B,C and D were extracted with phosphate buffer saline(PBS)or with 70%EtOH at pH values 8.0,7.2 and 6.4 followed by:(1)reverse-phase high-performance liquid chromatography(RP-HPLC)on Purospher®Star RP-18 column,the quantity of caffeic acid,chrysin,pinocembrin and galangin was determined;(2)determination of total flavonoids in both extracts;(3)antimicrobial tests of both extracts against(a)Gram-positive bacteria:methicillin-resistant Staphylococcus aureus(MRSA),St.aureus,Streptococcus pyogenes,Str.agalactiae,(b)Gram-negative bacteria:Escherichia coli,Pseudomonas aeruginosa,Proteus mirabilis,Acinetobacter baumanii and(c)yeast:Candida albicans.The antimicrobial activity of propolis’PBS extracts against Gram-positive bacteria shows the lowest minimal inhibitory concentration(MIC,mg/mL)at pH 8.0 in sample C,followed by A,B and D.In sample C,MICs at pH 8.0 were 0.007(Str.agalactiae),0.015(MRSA),0.015(Str.pyogenes)and 0.007(St.aureus).The polyphenol content of sample C is:flavonoid content 5.47±0.62 mg/mL,caffeic acid 1.33±0.92 mg/mL,chrysin 41.02±4.22μg/mL,pinocembrin 2.93±0.33 mg/mL and galangin 41.87±4.23 mg/mL.PBS extracts against Gram-negative bacteria show the lowest MIC(mg/mL)at pH 8.0 in sample D,followed by B,C and A.In sample D,MICs at pH 8.0 were 0.003(Acin.baumanii,Pr.mirabilis,Ps.aeruginosa)and 0.007(E.coli).Polyphenol content of sample D is:flavonoids 8.28±0.92 mg/mL,caffeic acid 3.56±0.32 mg/mL,chrysin 677.42±68.42μg/mL,pinocembrin 146.49±13.89 mg/mL and galangin 59.81±5.86 mg/mL.The strongest anti C.albicans activity,with the lowest MIC(mg/mL),at pH 8.0 was in the sample C,followed by samples D,A and B.In sample C,the MIC at pH 8.0 is 0.001(PBS extract).The antimicrobial activities of selected propolis samples correlate with their polyphenol content,more precisely,flavonoid,caffeic acid,chrysin,pinocembrin and galangin content.展开更多
文摘Extraction of proteins from pine needles for proteomic analysis has long been a challenge for scientists. We compared three different protein extraction methods including sucrose, Tris-HCl and trichloroacetic acid （TCA）/acetone （TCA method） to determine their efficiency in separating pine needle proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis （SDS-PAGE） and two-dimensional PAGE （2D-PAGE）. Proteins were then separated by SDS-PAGE. Among three methods the method using sucrose extraction buffer showed the highest efficiency and highest quality in separating proteins. In addition, clearer and more stable strips were detected by SDS-PAGE using sucrose extraction buffer. When the proteins extracted using sucrose extraction buffer were separated by 2D-PAGE, more than 300 protein spots, with isoelectric points （PI） ranging from 4.0 to 7,0 and molecular weights （MW） from 6.5 to 97.4 kD, were observed. This confirmed that the method with sucrose extraction buffer was an efficient and reliable method for extracting proteins from pine needles.
基金Project supported by Punjab Agricultural Research Board (PARB)the project No.150 awarded to Dr.Iqrar Ahmad KHAN,Pakistan
文摘Good quality deoxyribonucleic acid （DNA） is the pre-requisite for its downstream applications. The presence of high concentrations of polysaccharides, polyphenols, proteins, and other secondary metabolites in mango leaves poses problem in getting good quality DNA fit for polymerase chain reaction （PCR） applications. The problem is exacerbated when DNA is extracted from mature mango leaves. A reliable and modified protocol based on the cetyl trimethylammonium bromide （CTAB） method for DNA extraction from mature mango leaves is described here. High concentrations of inert salt were used to remove polysaccharides; Polyvinylpyrrolidone （PVP） and 13-mercaptoethanol were employed to man＇age phenolic compounds. Extended chloroform-isoamyl alcohol treatment followed by RNase treatment yielded 950-1050 pg of good quality DNA, free of protein and RNA. The problems of DNA degradation,contamination, and low yield due to irreversible binding of phenolic compounds and coprecipitation of polysaccharides with DNA were avoided by this method, The DNA isolated by the modified method showed good PCR amplification using simple sequence repeat （SSR） primers, This modified protocol can also be used to extract DNA from other woody plants having similar problems.
基金supported by the National Natural Science Foundation of China(Nos.41401565,41201520 and 20807046)
文摘Bioavailability is a critical factor for assessing the environmental risk of organic pollutants in soil. In this study, extractions with 3 different solvents, including 2 aqueous solutions, calcium chloride(CaCl_2) and a phosphate buffer solution(PBS), and a mixture of aqueous solution and organic solvent, a PBS-methanol(8:2,volume/volume) mixture(PBS-M), were performed to assess the bioavailability of chlorimuron-ethyl in soil in comparison to a battery of toxicity tests in wheat seedlings. The results indicated that the peroxidase(POD) activity in wheat leaves after 7 d of exposure was one of the sensitive biomarkers of chlorimuron-ethyl in soil.The extractability of chlorimuron-ethyl by all the 3 solvents decreased with exposure time, and the rate of decrease of the PBS-M extraction between 1 and 7 d of exposure was substantially higher than those of the aqueous solution extractions. Chlorimuron-ethyl gradually changed from a water-soluble form into a soil organic matter(SOM)-bound form in the soil. The PBS extraction correlated best with the POD activity in the leaves after 7 d of exposure.
基金the frame of Croatian Institute for Experimental and Translational Oncology(CIETO)supported by IvanČermak from the Crodux-plin,Savska Opatovina,10000 Zagreb,Croatia。
文摘Propolis is a resinous natural product,produced by bees(Apis mellifera),from vegetable parts and plant secretions.Propolis’samples A,B,C and D were extracted with phosphate buffer saline(PBS)or with 70%EtOH at pH values 8.0,7.2 and 6.4 followed by:(1)reverse-phase high-performance liquid chromatography(RP-HPLC)on Purospher®Star RP-18 column,the quantity of caffeic acid,chrysin,pinocembrin and galangin was determined;(2)determination of total flavonoids in both extracts;(3)antimicrobial tests of both extracts against(a)Gram-positive bacteria:methicillin-resistant Staphylococcus aureus(MRSA),St.aureus,Streptococcus pyogenes,Str.agalactiae,(b)Gram-negative bacteria:Escherichia coli,Pseudomonas aeruginosa,Proteus mirabilis,Acinetobacter baumanii and(c)yeast:Candida albicans.The antimicrobial activity of propolis’PBS extracts against Gram-positive bacteria shows the lowest minimal inhibitory concentration(MIC,mg/mL)at pH 8.0 in sample C,followed by A,B and D.In sample C,MICs at pH 8.0 were 0.007(Str.agalactiae),0.015(MRSA),0.015(Str.pyogenes)and 0.007(St.aureus).The polyphenol content of sample C is:flavonoid content 5.47±0.62 mg/mL,caffeic acid 1.33±0.92 mg/mL,chrysin 41.02±4.22μg/mL,pinocembrin 2.93±0.33 mg/mL and galangin 41.87±4.23 mg/mL.PBS extracts against Gram-negative bacteria show the lowest MIC(mg/mL)at pH 8.0 in sample D,followed by B,C and A.In sample D,MICs at pH 8.0 were 0.003(Acin.baumanii,Pr.mirabilis,Ps.aeruginosa)and 0.007(E.coli).Polyphenol content of sample D is:flavonoids 8.28±0.92 mg/mL,caffeic acid 3.56±0.32 mg/mL,chrysin 677.42±68.42μg/mL,pinocembrin 146.49±13.89 mg/mL and galangin 59.81±5.86 mg/mL.The strongest anti C.albicans activity,with the lowest MIC(mg/mL),at pH 8.0 was in the sample C,followed by samples D,A and B.In sample C,the MIC at pH 8.0 is 0.001(PBS extract).The antimicrobial activities of selected propolis samples correlate with their polyphenol content,more precisely,flavonoid,caffeic acid,chrysin,pinocembrin and galangin content.
