This manuscript was edited by Wallace Academic Editing.

1. Preparation of Ethanol-extracted Compounds
<ol>
	<li>Weigh 10 g of frozen Taiwanese green propolis, which was collected from beehives in Taiwan from May to July, and grind it using the spice grinder. Confirm that whole pieces of Taiwanese green propolis are ground into a fine powder without any large particles.</li>
	<li>Add 100 mL of various concentrations of ethanol (60%, 70%, 80%, 95%, and 99.5%) and water to separate flasks and mix each concentration with 10 g of ground propolis.</li>
	<li>Incubate at 25 &#176;C and shake the flask at 250 rpm for 48 h.</li>
	<li>Filter the ethanol extracts through filter paper with a 25 &#956;m pore size.</li>
	<li>Reconstitute the filtrates to their original volume (100 mL) with 95% ethanol using a volumetric flask.</li>
	<li>Store the ethanol extracts at -20 &#176;C. 
	NOTE: The protocol can be paused here.</li>
</ol>
2. Preparation of Ethanol Extracts for HPLC
<ol>
	<li>Concentrate 10 mL of ethanol extracts by vacuum evaporation at 40 &#176;C for 15 min.</li>
	<li>Bake the dry matter at 45 &#176;C for 24 h.</li>
	<li>Reconstitute the dry matter with 10 mL of 95% ethanol.</li>
	<li>Filter 1 mL of ethanol extracts using a sterile syringe filter with a 0.45 &#181;m pore size.</li>
	<li>Refilter the ethanol extracts using a sterile syringe filter with a 0.22 &#181;m pore size. The filtrate is collected and can be directly analyzed using HPLC.</li>
</ol>
3. Analysis of the Propolin Content Using HPLC
<ol>
	<li>Establishment of standard curves of propolins
	<ol>
		<li>Prepare 1 L of the mobile phase of 88.8:11.2 (v/v) methanol:water solution.</li>
		<li>Prepare serial dilutions of propolin standard (C, D, F, and G) concentrations (15.625 mg/mL, 31.25 mg/mL, 62.5 mg/mL, and 125 mg/mL, respectively) using the mobile phase solution as a solvent.</li>
		<li>Inject 20 &#181;L of propolin standard concentrations into the reverse-phase column, sequentially from the low concentration to the high concentration.</li>
		<li>Set the HPLC column at 30 &#176;C and the flow rate to 1 mL/min.</li>
		<li>Set the wavelength of the UV detector to 280 nm and the recorder time to 20 min.</li>
		<li>Analyze the standards at least 3x.</li>
		<li>Plot the measurement response (y-axis) against the concentration (x-axis) using calculation sheet software, and create a standard curve with the equation and R-square value.</li>
	</ol>
	</li>
	<li>Analysis of ethanol extracts
	<ol>
		<li>Inject 20 &#181;L of the ethanol extracts obtained from step 2.5 into the reverse-phase column.</li>
		<li>Set the HPLC column at 30 &#176;C and the flow rate to 1 mL/min.</li>
		<li>Set the wavelength of the UV detector to 280 nm and the recorder time to 20 min.</li>
		<li>Analyze the standards at least 3x.</li>
		<li>Calculate the concentration of propolin in the ethanol extract, using the equation for the standard curve obtained from step 3.1.7 that uses the peak area for each propolin.</li>
	</ol>
	</li>
</ol>
4. Minimum Inhibitory Concentration and Minimum Bactericidal Concentration Analysis
NOTE: The microdilution method is used to evaluate the antibacterial efficacy of ethanol-extracted propolins from Taiwanese green propolis.
<ol>
	<li>Preparation of test organisms
	<ol>
		<li>Thaw the bacterial strains, Staphylococcus aureus and Escherichia coli, and then, culture them in tryptic soy broth and nutrient broth, respectively, at 37 &#176;C for 24 h.</li>
		<li>Passage the S. aureus using tryptic soy broth and E. coli using nutrient broth for two generations and, then, calculate colony-forming units by counting individual colonies on an agar plate.</li>
	</ol>
	</li>
	<li>Preparation of ethanol extracts for antibacterial activity testing
	<ol>
		<li>Concentrate the ethanol extracts obtained from step 1.5 by vacuum evaporation at 40 &#176;C for 15 min.</li>
		<li>Reconstitute the dry matter with dimethyl sulfoxide (DMSO) and adjust the concentration of the extract to 12.8 mg/mL.</li>
		<li>Make a serial dilution (concentrations: 5, 10, 20, 40, 80, 160, 320, and 640 &#181;g/mL) of ethanol extracts using the broth.</li>
	</ol>
	</li>
	<li>Minimum inhibitory concentration tests
	<ol>
		<li>Add 10 &#181;L of diluted ethanol extracts ranging from 0.156 to 640.0 &#181;g/mL into a 96-well plate.</li>
		<li>Using broth, adjust the volume to 100 &#181;L, and maintain 5% DMSO in all the dilutions.</li>
		<li>Inoculate 100 &#181;L of bacterial culture (1 x 106/mL) into the 96-well plate.</li>
		<li>Culture inoculum containing various concentrations of ethanol extracts at 37 &#176;C for 48 h.</li>
		<li>Analyze the bacterial growth according to turbidity and using optical density (microplate reader) at 590 nm to determine the minimum inhibitory concentration (MIC).</li>
	</ol>
	</li>
	<li>Minimum bactericidal concentration tests
	<ol>
		<li>Inoculate 10 &#181;L of liquid culture from each well of the MIC test that exhibited no growth onto an agar plate.</li>
		<li>Incubate at 37 &#176;C for 24 h.</li>
		<li>Determine the bactericidal activity by identifying the lowest concentration that revealed no visible bacterial growth. The concentration that completely eliminates cell growth is considered to be the minimum bacterial concentration (MBC).</li>
	</ol>
	</li>
</ol>

<ol>
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K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+radical+scavenging+activity,+cytotoxic+effects+and+apoptosis+induction+in+human+melanoma+cells+by+Taiwanese+propolis+from+different+sources.">Comparison of radical scavenging activity, cytotoxic effects and apoptosis induction in human melanoma cells by Taiwanese propolis from different sources.</a> Evidence-Based Complementary and Alternative. 1 (2), 175-185 (2004).</li><li>Chen, C. N., Wu, C. L., Lin, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Apoptosis+of+human+melanoma+cells+induced+by+the+novel+compounds+propolin+A+and+propolin+B+from+Taiwanese+propolis.">Apoptosis of human melanoma cells induced by the novel compounds propolin A and propolin B from Taiwanese propolis.</a> Cancer Letters. 24 (1-2), 218-231 (2007).</li><li>Huang, W. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Propolin+G,+a+prenylflavanone,+isolated+from+Taiwanese+propolis,+induces+caspase-dependent+apoptosis+in+brain+cancer+cells.">Propolin G, a prenylflavanone, isolated from Taiwanese propolis, induces caspase-dependent apoptosis in brain cancer cells.</a> Journal of Agricultural and Food Chemistry. 55 (18), 7366-7376 (2007).</li><li>Chen, Y. W., Ye, S. R., Ting, C., Yu, Y. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibacterial+activity+of+propolins+from+Taiwanese+green+propolis.">Antibacterial activity of propolins from Taiwanese green propolis.</a> Journal of Food and Drug Analysis. 26 (2), 761-768 (2018).</li><li>Hegazi, A. G., Abd El Hady, F. K., Abd Allah, F. A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+composition+and+antimicrobial+activity+of+European+propolis.">Chemical composition and antimicrobial activity of European propolis.</a> Zeitschrift f&#252;r Naturforschung. 55 (1-2), 70-75 (2000).</li><li>Sforcin, J. M., Fernandes, A., Lopes, C. A. M., Bankova, V., Funari, S. R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Seasonal+effect+on+Brazilian+propolis+antibacterial+activity.">Seasonal effect on Brazilian propolis antibacterial activity.</a> Journal of Ethnopharmacology. 73 (1-2), 243-249 (2000).</li><li>Chen, Y. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+Taiwanese+propolis+collected+from+different+locations+and+seasons.">Characterization of Taiwanese propolis collected from different locations and seasons.</a> Journal of the Science of Food and Agriculture. 88 (3), 412-419 (2008).</li><li>Grange, J. M., Davey, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibacterial+properties+of+propolis+(bee+glue).">Antibacterial properties of propolis (bee glue).</a> Journal of the Royal Society of Medicine. 83 (3), 159-160 (1990).</li><li>Kujumgiev, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibacterial,+antifungal+and+antiviral+activity+of+propolis+from+different+geographic+origins.">Antibacterial, antifungal and antiviral activity of propolis from different geographic origins.</a> Journal of Ethnopharmacology. 64 (3), 235-240 (1999).</li><li>Krol, W., Scheller, S., Shani, J., Pietsz, G., Czuba, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synergistic+effect+of+ethanol+extract+of+propolis+and+antibiotics+in+the+growth+of+Staphylococcus+aureus.">Synergistic effect of ethanol extract of propolis and antibiotics in the growth of Staphylococcus aureus.</a> Drug Research. 43 (5), 607-609 (1993).</li><li>Lu, L. C., Chen, Y. W., Chou, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibacterial+and+DPPH+free+radical-scavenging+activities+of+the+ethanol+extract+of+propolis+collected+in+Taiwan.">Antibacterial and DPPH free radical-scavenging activities of the ethanol extract of propolis collected in Taiwan.</a> Journal of Food and Drug Analysis. 11 (4), 277-282 (2003).</li><li>Tosi, B., Donini, A., Romagnoli, C., Bruni, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antimicrobial+activity+of+some+commercial+extracts+of+propolis+prepared+with+different+solvents.">Antimicrobial activity of some commercial extracts of propolis prepared with different solvents.</a> Phytotherapy Research. 10 (4), 335-336 (1996).</li><li>Cunha, I. B. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Factors+that+influence+the+yield+and+composition+of+Brazilian+propolis+extracts.">Factors that influence the yield and composition of Brazilian propolis extracts.</a> Journal of the Brazilian Chemical Society. 15 (6), 964-970 (2004).</li><li>Woo, S. O., Hong, I. P., Han, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extraction+properties+of+propolis+with+ethanol+concentration.">Extraction properties of propolis with ethanol concentration.</a> Journal of Apicultural Research. 30 (3), 211-216 (2015).</li><li>Park, Y. K., Ikegaki, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+water+and+ethanolic+extracts+of+propolis+and+evaluation+of+the+preparations.">Preparation of water and ethanolic extracts of propolis and evaluation of the preparations.</a> Bioscience, Biotechnology, and Biochemistry. 62 (11), 2230-2232 (1998).</li><li>Ramanauskien&#279;, K., Ink&#279;nien&#279;, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Propolis+oil+extract:+quality+analysis+and+evaluation+of+its+antimicrobial+activity.">Propolis oil extract: quality analysis and evaluation of its antimicrobial activity.</a> Natural Product Research. 25 (15), 1463-1468 (2011).</li><li>Machado, B. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+composition+and+biological+activity+of+extracts+obtained+by+supercritical+extraction+and+ethanolic+extraction+of+brown,+green+and+red+propolis+derived+from+different+geographic+regions+in+brazil.">Chemical composition and biological activity of extracts obtained by supercritical extraction and ethanolic extraction of brown, green and red propolis derived from different geographic regions in brazil.</a> PLoS One. 11 (1), e0145954 (2016).</li><li>Monroy, Y. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brazilian+green+propolis+extracts+obtained+by+conventional+processes+and+by+processes+at+high+pressure+with+supercritical+carbon+dioxide,+ethanol+and+water.">Brazilian green propolis extracts obtained by conventional processes and by processes at high pressure with supercritical carbon dioxide, ethanol and water.</a> The Journal of Supercritical Fluids. 130, 189-197 (2017).</li><li>Popova, M., Chen, C. N., Chen, P. Y., Huang, C. 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The authors have nothing to declare.

A study reported that maceration, using various concentrations of ethanol, could be used for Brazilian propolis extraction17; however, the process was time-consuming17. It took at least 10 days to extract the bioactive compounds, such as phenolic content, from Brazilian propolis17. Alternatively, ethanol extraction in combination with heating at 37 &#176;C, 50 &#176;C, or 70 &#176;C for 30 min has been proposed for extracting Brazilian propolis<sup c...

Propolis is a natural resinous mixture produced by the bee species Apis mellifera. Propolis has been widely used since ancient times in folk medicines. A study recently reported that propolis is beneficial for preventing microbial infections and inflammation1. Numerous studies have demonstrated that the main bioactive compounds in propolis are flavonoids, phenolic acid esters, prenylated p-coumaric acids, and diterpenic acids2,3. To date, 10 prenylated flavanone derivatives from Taiwanese green propolis have been identified through high-performance liquid chromatography (HPLC)4,5,6,7. The most abundant among these are propolins C, D, F, and G5,7. The considerable biological effects of Taiwanese green propolis are correlated with its high content of propolins8.
The concentrations of bioactive compounds in propolis vary greatly depending on the season and geographic location from which the propolis is obtained. European propolis mainly contains the flavonoid aglycone and phenolic acids9. The major bioactive compounds in propolis from Brazil are prenylated p-coumaric acids, such as artepillin C10. A study demonstrated that the season is a critical factor for determining the total propolin content in Taiwanese green propolis11. The propolin content in Taiwanese green propolis is highest in summer (May - July) and lowest in winter11. The antibacterial property of propolis has been widely considered to be an indicator of biological activity. Generally, the samples of propolis collected from various regions have exhibited a similar antibacterial property; for example, it is generally effective against almost all gram-positive bacteria and exhibits a limited antibacterial effect against gram-negative bacteria10,12,13. Synergistic interactions between the flavonoids in propolis were demonstrated to have an antibacterial effect14. Similarly, Taiwanese green propolis was reported to have an antimicrobial effect against gram-positive bacteria15. Furthermore, a study also identified an antimicrobial effect from the interactions of propolins in Taiwanese green propolis8.
The characterization of the bioactive compounds in propolis is difficult because its chemical composition can vary according to its source of origin. Therefore, it is necessary to establish a feasible and repeatable method for determining the quality of the Taiwanese green propolis. However, no standard procedure has been established for Taiwanese green propolis extraction and subsequent functional analysis. Several methods that variously apply organic and inorganic solvents have been proposed for propolis extraction16,17,18,19,20. Because propolis is a lipophilic mixture, studies have demonstrated that organic extraction is better than inorganic extraction8,18,19. The total propolin concentration in Taiwanese green propolis and its antibacterial properties are key indicators of the quality of Taiwanese green propolis. Thus, the purpose of this study is to present a protocol for using ethanol as a solvent to extract and characterize the antibacterial properties of Taiwanese green propolis.

<table>
	<tbody>
		<tr>
			<td>ethanol</td>
			<td>Sigma-Aldrich</td>
			<td>E7023</td>
			<td></td>
		</tr>
		<tr>
			<td>Whatman no. 4 filter paper</td>
			<td>Sigma-Aldrich</td>
			<td>WHA1004125</td>
			<td></td>
		</tr>
		<tr>
			<td>methanol</td>
			<td>Sigma-Aldrich</td>
			<td>34860</td>
			<td></td>
		</tr>
		<tr>
			<td>reverse phase RP-18 column</td>
			<td>Phenomenex Inc.</td>
			<td>00G-0234-E0</td>
			<td></td>
		</tr>
		<tr>
			<td>Staphylococcus aureus</td>
			<td>ATCC</td>
			<td>BCRC 10780</td>
			<td></td>
		</tr>
		<tr>
			<td>Escherichia coli</td>
			<td>ATCC</td>
			<td>BCRC 10675</td>
			<td></td>
		</tr>
		<tr>
			<td>tryptic soy broth</td>
			<td>Sigma-Aldrich</td>
			<td>22092</td>
			<td></td>
		</tr>
		<tr>
			<td>nutrient broth</td>
			<td>Sigma-Aldrich</td>
			<td>70122</td>
			<td></td>
		</tr>
		<tr>
			<td>dimethyl sulfoxide</td>
			<td>Sigma-Aldrich</td>
			<td>D2650</td>
			<td></td>
		</tr>
		<tr>
			<td>spice grinder</td>
			<td>Waring</td>
			<td>WSG60K</td>
			<td></td>
		</tr>
		<tr>
			<td>microplate Reader</td>
			<td>Molecular Devices</td>
			<td>EMAX PLUS</td>
			<td></td>
		</tr>
		<tr>
			<td>HPLC system</td>
			<td>Agilent</td>
			<td>1200 Series</td>
			<td></td>
		</tr>
		<tr>
			<td>vacuum evaporation</td>
			<td>B&#220;CHI</td>
			<td>Rotavapor R-215</td>
			<td></td>
		</tr>
	</tbody>
</table>

extraction and analysis of taiwanese green propolis

Taiwanese green propolis is rich in prenylated flavonoids and exhibits a broad range of biological activities, such as antioxidant, antibacterial, and anticancer ones. The bioactive compounds of Taiwanese green propolis are propolins, namely C, D, F, and G. The concentration of propolins in Taiwanese green propolis varies depending on the season and geographic location. Thus, it is critical to establish a standard and repeatable procedure for determining the quality of Taiwanese green propolis. Here, we present a protocol that uses ethanol-based extraction, high-performance liquid chromatography, and an antibacterial activity analysis to characterize Taiwanese green propolis quality. This method indicates that 95% and 99.5% ethanol extractions achieve the maximum dry matter yields from Taiwanese green propolis, thereby yielding the highest concentrations of propolins that have antibacterial properties. According to these findings, the present protocol is deemed reliable and repeatable for determining the quality of Taiwanese green propolis.

Positive representative data for the ethanol extraction are presented in Table 1. The dry matter yield from Taiwanese green propolis was positively associated with the concentration of ethanol. The 95% and 99.5% ethanol extracts had the highest dry matter yield from Taiwanese green propolis. The lowest dry matter yield from Taiwanese green propolis occurred when water was used as the extraction solvent. These results indicate that an organic solvent, such as ethanol, perf...

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Extraction and Analysis of Taiwanese Green Propolis

We present a protocol for using ethanol as a solvent to extract and characterize Taiwanese green propolis that exhibits antibacterial activity.

Taiwanese green propolis is rich in prenylated flavonoids and exhibits a broad range of biological activities, such as antioxidant, antibacterial, and ...

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8.4K Views.  National Ilan University. We present a protocol for using ethanol as a solvent to extract and characterize Taiwanese green propolis that exhibits antibacterial activity.

Video: Extraction and Analysis of Taiwanese Green Propolis

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Analysis of the cellular proteome can help to elucidate the molecular mechanisms underlying diseases due to the development of technologies that permit the large-scale identification and quantification of the proteins present in complex biological systems.The knowledge gained from a proteomic approach can potentially lead to a better understanding of the pathogenic mechanisms underlying diseases, allowing for the identification of novel diagnostic and prognostic disease markers, and, hopefully, of therapeutic targets. However, the cardiac mitral valve represents a very challenging sample for proteomic analysis because of the low cellularity in proteoglycan and collagen-enriched extracellular matrix. This makes it challenging to extract proteins for a global proteomic analysis. This work describes a protocol that is compatible with subsequent protein analysis, such as quantitative proteomics and immunoblotting. This can allow for the correlation of data concerning protein expression with data on quantitative mRNA expression and non-quantitative immunohistochemical analysis. Indeed, these approaches, when performed together, will lead to a more comprehensive understanding of the molecular mechanisms underlying diseases, from mRNA to post-translational protein modification. Thus, this method can be relevant to researchers interested in the study of cardiac valve physiopathology.

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Analysis of the cellular proteome can help to elucidate the molecular mechanisms underlying diseases due to the development of technologies that permit ...

A Simple Fractionated Extraction Method for the Comprehensive Analysis of Metabolites, Lipids, and Proteins from a Single Sample

Understanding of complex biological systems requires the measurement, analysis and integration of multiple compound classes of the living cell, usually determined by transcriptomic, proteomic, metabolomics and lipidomic measurements. In this protocol, we introduce a simple method for the reproducible extraction of metabolites, lipids and proteins from biological tissues using a single aliquot per sample. The extraction method is based on a methyl tert-butyl ether: methanol: water system for liquid: liquid partitioning of hydrophobic and polar metabolites into two immiscible phases along with the precipitation of proteins and other macromolecules as a solid pellet. This method, therefore, provides three different fractions of specific molecular composition, which are fully compatible with common high throughput 'omics' technologies such as liquid chromatography (LC) or gas chromatography (GC) coupled to mass spectrometers. Even though the method was initially developed for the analysis of different plant tissue samples, it has proved to be fully compatible for the extraction and analysis of biological samples from systems as diverse as algae, insects, and mammalian tissues and cell cultures.

A protocol for comprehensive extraction of lipids, metabolites and proteins from biological tissues using one sample is presented.

Understanding of complex biological systems requires the measurement, analysis and integration of multiple compound classes of the living cell, usually ...

Caffeine Extraction, Enzymatic Activity and Gene Expression of Caffeine Synthase from Plant Cell Suspensions

Caffeine (1,3,7-trimethylxanthine) is a purine alkaloid present in popular drinks such as coffee and tea. This secondary metabolite is regarded as a chemical defense because it has antimicrobial activity and is considered a natural insecticide. Caffeine can also produce negative allelopathic effects that prevent the growth of surrounding plants. In addition, people around the world consume caffeine for its analgesic and stimulatory effects. Due to interest in the technological applications of caffeine, research on the biosynthetic pathway of this compound has grown. These studies have primarily focused on understanding the biochemical and molecular mechanisms that regulate the biosynthesis of caffeine. In vitro tissue culture has become a useful system for studying this biosynthetic pathway. This article will describe a step-by-step protocol for the quantification of caffeine and for measuring the transcript levels of the gene (CCS1) encoding caffeine synthase (CS) in cell suspensions of C. arabica L. as well as its activity.

This protocol describes an efficient methodology for the extraction and quantification of caffeine in cell suspensions of C. arabica L. and an experimental process for evaluating the enzymatic activity of caffeine synthase with the expression level of the gene that encodes this enzyme.

Caffeine (1,3,7-trimethylxanthine) is a purine alkaloid present in popular drinks such as coffee and tea. This secondary metabolite is regarded as a ...

Characterization of Membrane Transporters by Heterologous Expression in E. coli and Production of Membrane Vesicles

Several methods have been developed to functionally characterize novel membrane transporters. Polyamines are ubiquitous in all organisms, but polyamine exchangers in plants have not been identified. Here, we outline a method to characterize polyamine antiporters using membrane vesicles generated from the lysis of Escherichia coli cells heterologously expressing a plant antiporter. First, we heterologously expressed AtBAT1 in an E. coli strain deficient in polyamine and arginine exchange transporters. Vesicles were produced using a French press, purified by ultracentrifugation and utilized in a membrane filtration assay of labeled substrates to demonstrate the substrate specificity of the transporter. These assays demonstrated that AtBAT1 is a proton-mediated transporter of arginine, &#947;-aminobutyric acid (GABA), putrescine and spermidine. The mutant strain that was developed for the assay of AtBAT1 may be useful for the functional analysis of other families of plant and animal polyamine exchangers. We also hypothesize that this approach can be used to characterize many other types of antiporters, as long as these proteins can be expressed in the bacterial cell membrane. E. coli is a good system for the characterization of novel transporters, since there are multiple methods that can be employed to mutagenize native transporters.

Characterization of Membrane Transporters by Heterologous Expression in E. coli and Production of Membrane Vesicles

We describe a method for the characterization of proton-driven membrane transporters in membrane vesicle preparations produced by heterologous expression in E. coli and lysis of cells using a French press.

Several methods have been developed to functionally characterize novel membrane transporters. Polyamines are ubiquitous in all organisms, but polyamine ...

Rapid and Cost-Effective RNA Extraction of Rat Pancreatic Tissue

Regardless of the extraction method, optimized RNA extraction of tissues and cell lines are carried out in four stages: 1) homogenization, 2) effective denaturation of proteins from RNA, 3) ribonuclease inactivation, and 4) removal of contamination from DNA, proteins, and carbohydrates. However, it is very laborious to maintain the integrity of RNA when there are high levels of RNase in the tissue. Spontaneous autolysis makes it very difficult to extract RNA from pancreatic tissue without damaging it. Thus, a practical RNA extraction method is needed to maintain the integrity of pancreatic tissues during the extraction process. An experimental and comparative study of existing protocols was carried out by obtaining 20-30 mg of rat pancreatic tissues in less than 2 minutes and extracting the RNA. The results were assessed by electrophoresis. The experiments were carried out three times for generalization of the results. Immersing pancreatic tissue in RNA stabilization reagent at -80 &#176;C for 24 h yielded high integrity RNA, when the RNA extraction reagent was used as the reagent. The results obtained were comparable to the results obtained from commercial kits with spin column bindings.

The purity and integrity of the isolated RNA is a vital step in RNA dependent assays. Here, we present a practical, rapid, and inexpensive method to extract RNA from a small quantity of undamaged pancreatic tissue.

Regardless of the extraction method, optimized RNA extraction of tissues and cell lines are carried out in four stages: 1) homogenization, 2) effective ...

Ganglioside Extraction, Purification and Profiling

Gangliosides are glycosphingolipids that contain one or more sialic acid residues. They are found on all vertebrate cells and tissues but are especially abundant in the brain. Expressed primarily on the outer leaflet of the plasma membranes of cells, they modulate the activities of cell surface proteins via lateral association, act as receptors in cell-cell interactions and are targets for pathogens and toxins. Genetic dysregulation of ganglioside biosynthesis in humans results in severe congenital nervous system disorders. Because of their amphipathic nature, extraction, purification, and analysis of gangliosides require techniques that have been optimized by many investigators in the 80 years since their discovery. Here, we describe bench-level methods for the extraction, purification, and preliminary qualitative and quantitative analyses of major gangliosides from tissues and cells that can be completed in a few hours. We also describe methods for larger scale isolation and purification of major ganglioside species from brain. Together, these methods provide analytical and preparative scale access to this class of bioactive molecules.

Gangliosides are sialic acid-bearing glycosphingolipids that are particularly abundant in the brain. Their amphipathic nature requires organic/aqueous extraction and purification techniques to ensure optimal recovery and accurate analyses. This article provides overviews of analytic and preparative scale ganglioside extraction, purification, and thin layer chromatography analysis.

Gangliosides are glycosphingolipids that contain one or more sialic acid residues. They are found on all vertebrate cells and tissues but are especially ...

Extraction and Visualization of Protein Aggregates after Treatment of Escherichia coli with a Proteotoxic Stressor

The exposure of living organisms to environmental and cellular stresses often causes disruptions in protein homeostasis and can result in protein aggregation. The accumulation of protein aggregates in bacterial cells can lead to significant alterations in the cellular phenotypic behavior, including a reduction in growth rates, stress resistance, and virulence. Several experimental procedures exist for the examination of these stressor-mediated phenotypes. This paper describes an optimized assay for the extraction and visualization of aggregated and soluble proteins from different Escherichia coli strains after treatment with a silver-ruthenium-containing antimicrobial. This compound is known to generate reactive oxygen species and causes widespread protein aggregation. 
The method combines a centrifugation-based separation of protein aggregates and soluble proteins from treated and untreated cells with subsequent separation and visualization by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Coomassie staining. This approach is simple, fast, and allows a qualitative comparison of protein aggregate formation in different E. coli strains. The methodology has a wide range of applications, including the possibility to investigate the impact of other proteotoxic antimicrobials on in vivo protein aggregation in a wide range of bacteria. Moreover, the protocol can be used to identify genes that contribute to increased resistance to proteotoxic substances. Gel bands can be used for the subsequent identification of proteins that are particularly prone to aggregation.

Extraction and Visualization of Protein Aggregates after Treatment of Escherichia coli with a Proteotoxic Stressor

This protocol describes the extraction and visualization of aggregated and soluble proteins from Escherichia coli after treatment with a proteotoxic antimicrobial. Following this procedure allows a qualitative comparison of protein aggregate formation in vivo in different bacterial strains and/or between treatments.

The exposure of living organisms to environmental and cellular stresses often causes disruptions in protein homeostasis and can result in protein ...

Ultrasonic-Assisted Extraction of Cannabidiolic Acid from Cannabis Biomass

Industrial hemp (Cannabis spp.) has many compounds of interest with potential medical benefits. Of these compounds, cannabinoids have come to the center of attention, specifically acidic cannabinoids. The focus is turning toward acidic cannabinoids due to their lack of psychotropic activity. Cannabis plants produce acidic cannabinoids with hemp plants producing low levels of psychotropic cannabinoids. As such, utilization of hemp for acidic cannabinoid extraction would eliminate the need for decarboxylation prior to extraction as a source for the cannabinoids. The use of solvent-based extraction is ideal for obtaining acidic cannabinoids as their solubility in solvents such as supercritical CO2 is limited due to the high pressure and temperature required to reach their solubility constants. An alternative method designed to increase solubility is ultrasonic-assisted extraction. In this protocol, the impact of solvent polarity (acetonitrile 0.46, ethanol 0.65, methanol 0.76, and water 1.00) and concentration (20%, 50%, 70%, 90%, and 100%) on ultrasonic-assisted extraction efficiency has been examined. Results show that water was the least effective and acetonitrile was the most effective solvent examined. Ethanol was further examined since it has the lowest toxicity and is generally regarded as safe (GRAS). Surprisingly, 50% ethanol in water is the most effective ethanol concentration for extracting the highest amount of cannabinoids from hemp. The increase in cannabidiolic acid concentration was 28% when compared to 100% ethanol, and 23% when compared to 100% acetonitrile. While it was determined that 50% ethanol is the most effective concentration for our application, the method has also been demonstrated to be effective with alternative solvents. Consequently, the proposed method is deemed effective and rapid for extracting acidic cannabinoids.

Ultrasonic-Assisted Extraction of Cannabidiolic Acid from Cannabis Biomass

Ultrasonic-assisted extraction (UAE) increases extraction efficiency of solvents and when applied to Cannabis spp. biomass it reduces the time required for extraction. This decreases the cost and potential cannabinoid loss due to degradation. Additionally, UAE is considered a green method due to low solvent use.

Industrial hemp (Cannabis spp.) has many compounds of interest with potential medical benefits. Of these compounds, cannabinoids have come to the center ...

The Extraction of Liver Glycogen Molecules for Glycogen Structure Determination

Liver glycogen is a hyperbranched glucose polymer that is involved in the maintenance of blood sugar levels in animals. The properties of glycogen are influenced by its structure. Hence, a suitable extraction method that isolates representative samples of glycogen is crucial to the study of this macromolecule. Compared to other extraction methods, a method that employs a sucrose density gradient centrifugation step can minimize molecular damage. Based on this method, a recent publication describes how the density of the sucrose solution used during centrifugation was varied (30%, 50%, 72.5%) to find the most suitable concentration to extract glycogen particles of a wide variety of sizes, limiting the loss of smaller particles. A 10 min boiling step was introduced to test its ability to denature glycogen degrading enzymes, thus preserving glycogen. The lowest sucrose concentration (30%) and the addition of the boiling step were shown to extract the most representative samples of glycogen.

An optimal sucrose concentration was determined for the extraction of liver glycogen using sucrose density gradient centrifugation. The addition of a 10 min boiling step to inhibit glycogen-degrading enzymes proved beneficial.

Liver glycogen is a hyperbranched glucose polymer that is involved in the maintenance of blood sugar levels in animals. The properties of glycogen are ...