We are thankful for the funding sources from the Division of Biology and Johnson Cancer Research Center for BRIEF and IRA awards, respectively to GV. We also thank the K-INBRE postdoctoral award to RV. This work was supported in part by the Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under grant number P20 GM103418. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institute of General Medical Sciences or the National Institutes of Health. We thank the anonymous reviewers for their helpful comments.

1. Setup and prerunning of standard liquid-phase IEF unit
<ol>
	<li>Assemble the liquid-phase IEF electrodes (anode-red button and cathode-black button) with their respective exchange membranes according to the instruction manual (see Table of Materials). Equilibrate the anion exchange membranes with 0.1 M NaOH and the cation exchange membranes with 0.1 M H3PO4 at least for 16 h when new membranes are used.
	<ol>
		<li>Store the membranes in electrolytes (0.1 M NaOH or 0.1 M H<sub...

<ol>
	<li>Jankowska, U., 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=Optimized+procedure+of+extraction,+purification+and+proteomic+analysis+of+nuclear+proteins+from+mouse+brain.">Optimized procedure of extraction, purification and proteomic analysis of nuclear proteins from mouse brain.</a> Journal of Neuroscience Methods. 261, 1-9 (2016).</li><li>Pergande, M. R., Cologna, 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=Isoelectric+Point+Separations+of+Peptides+and+Proteins.">Isoelectric Point Separations of Peptides and Proteins.</a> Proteomes. 5 (1), (2017).</li><li>Stoyanov, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IEF-based+multidimensional+applications+in+proteomics:+toward+higher+resolution.">IEF-based multidimensional applications in proteomics: toward higher resolution.</a> Electrophoresis. 33 (22), 3281-3290 (2012).</li><li>Vesterberg, O. A. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Method of Isoelectric Fractionation. , (1964).</li><li>Vesterberg, O., Svensson, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isoelectric+fractionation,+analysis,+and+characterization+of+ampholytes+in+natural+pH+gradients.+IV.+Further+studies+on+the+resolving+power+in+connection+with+separation+of+myoglobins.">Isoelectric fractionation, analysis, and characterization of ampholytes in natural pH gradients. IV. Further studies on the resolving power in connection with separation of myoglobins.</a> Acta Chemica Scandinavica. 20 (3), 820-834 (1966).</li><li>Righetti, P. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Isoelectric Focusing: Theory, Methodology and Applications. , 1 (1983).</li><li>Vesterberg, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+and+Isoelectric+Fractionation+of+Carrier+Ampholytes.">Synthesis and Isoelectric Fractionation of Carrier Ampholytes.</a> Acta Chemica Scandinavica. 23, 2653-2666 (1969).</li><li>Bier, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recycling+isoelectric+focusing+and+isotachophoresis.">Recycling isoelectric focusing and isotachophoresis.</a> Electrophoresis. 19 (7), 1057-1063 (1998).</li><li>Bier, M., Palusinski, O. A., Mosher, R. A., Saville, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrophoresis:+mathematical+modeling+and+computer+simulation.">Electrophoresis: mathematical modeling and computer simulation.</a> Science. 219 (4590), 1281-1287 (1983).</li><li>Ayala, A., Parrado, J., Machado, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+Rotofor+preparative+isoelectrofocusing+cell+in+protein+purification+procedure.">Use of Rotofor preparative isoelectrofocusing cell in protein purification procedure.</a> Applied Biochemistry and Biotechnology. 69 (1), 11-16 (1998).</li><li>Wagner, L., 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=Isolation+of+dipeptidyl+peptidase+IV+(DP+4)+isoforms+from+porcine+kidney+by+preparative+isoelectric+focusing+to+improve+crystallization.">Isolation of dipeptidyl peptidase IV (DP 4) isoforms from porcine kidney by preparative isoelectric focusing to improve crystallization.</a> Biological Chemistry. 392 (7), 665-677 (2011).</li><li>Hosken, B. D., Li, C., Mullappally, B., Co, C., Zhang, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+Characterization+of+Monoclonal+Antibody+Charge+Variants+by+Free+Flow+Isoelectric+Focusing.">Isolation and Characterization of Monoclonal Antibody Charge Variants by Free Flow Isoelectric Focusing.</a> Analytical Chemistry. 88 (11), 5662-5669 (2016).</li><li>Yu, J. 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=Francisella+tularensis+T-cell+antigen+identification+using+humanized+HLA-DR4+transgenic+mice.">Francisella tularensis T-cell antigen identification using humanized HLA-DR4 transgenic mice.</a> Clinical Vaccine Immunology. 17 (2), 215-222 (2010).</li><li>Riyong, D., 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=Size+and+charge+antigens+of+Dirofilaria+immitis+adult+worm+for+IgG-ELISA+diagnosis+of+bancroftian+filariasis.">Size and charge antigens of Dirofilaria immitis adult worm for IgG-ELISA diagnosis of bancroftian filariasis.</a> Southeast Asian Journal of Tropical Medicine and Public Health. 41 (2), 285-297 (2010).</li><li>Vediyappan, G., Bikandi, J., Braley, R., Chaffin, W. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+surface+proteins+of+Candida+albicans:+preparation+of+extracts+and+improved+detection+of+proteins.">Cell surface proteins of Candida albicans: preparation of extracts and improved detection of proteins.</a> Electrophoresis. 21 (5), 956-961 (2000).</li><li>Vediyappan, G., Dumontet, V., Pelissier, F., d'Enfert, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gymnemic+acids+inhibit+hyphal+growth+and+virulence+in+Candida+albicans.">Gymnemic acids inhibit hyphal growth and virulence in Candida albicans.</a> PLoS One. 8 (9), 74189 (2013).</li><li>Bradford, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+and+sensitive+method+for+the+quantitation+of+microgram+quantities+of+protein+utilizing+the+principle+of+protein-dye+binding.">A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.</a> Analytical Biochemistry. 72, 248-254 (1976).</li><li>Laemmli, U. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cleavage+of+structural+proteins+during+the+assembly+of+the+head+of+bacteriophage+T4.">Cleavage of structural proteins during the assembly of the head of bacteriophage T4.</a> Nature. 227 (5259), 680-685 (1970).</li><li>Riazi, S., Dover, S., Turovskiy, Y., Chikindas, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Commercial+ampholytes+used+for+isoelectric+focusing+may+interfere+with+bioactivity+based+purification+of+antimicrobial+peptides.">Commercial ampholytes used for isoelectric focusing may interfere with bioactivity based purification of antimicrobial peptides.</a> Journal of Microbiological Methods. 71 (1), 87-89 (2007).</li><li>Kamei, K., Takano, R., Miyasaka, A., Imoto, T., Hara, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amino-Acid-Sequence+of+Sweet-Taste-Suppressing+Peptide+(Gurmarin)+from+the+Leaves+of+Gymnema-Sylvestre.">Amino-Acid-Sequence of Sweet-Taste-Suppressing Peptide (Gurmarin) from the Leaves of Gymnema-Sylvestre.</a> Journal of Biochemistry. 111 (1), 109-112 (1992).</li><li>Craik, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemistry.+Seamless+proteins+tie+up+their+loose+ends.">Chemistry. Seamless proteins tie up their loose ends.</a> Science. 311 (5767), 1563-1564 (2006).</li><li>Craik, D. J., Daly, N. L., Bond, T., Waine, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plant+cyclotides:+A+unique+family+of+cyclic+and+knotted+proteins+that+defines+the+cyclic+cystine+knot+structural+motif.">Plant cyclotides: A unique family of cyclic and knotted proteins that defines the cyclic cystine knot structural motif.</a> Journal of Molecular Biology. 294 (5), 1327-1336 (1999).</li><li>Stoecklin, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemistry+and+physiological+properties+of+gymnemic+acid,+the+antisaccharine+principle+of+the+leaves+of+Gymnema+sylvestre.">Chemistry and physiological properties of gymnemic acid, the antisaccharine principle of the leaves of Gymnema sylvestre.</a> Journal of Agricultural and Food Chemistry. 17 (4), 704-708 (1969).</li><li>Liu, H. M., Kiuchi, F., Tsuda, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+structure+elucidation+of+gymnemic+acids,+antisweet+principles+of+Gymnema+sylvestre.">Isolation and structure elucidation of gymnemic acids, antisweet principles of Gymnema sylvestre.</a> Chemical &amp; Pharmaceutical Bulletin (Tokyo). 40 (6), 1366-1375 (1992).</li><li>Veerapandian, R., Vediyappan, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gymnemic+Acids+Inhibit+Adhesive+Nanofibrillar+Mediated+Streptococcus+gordonii-Candida+albicans+Mono-Species+and+Dual-Species+Biofilms.">Gymnemic Acids Inhibit Adhesive Nanofibrillar Mediated Streptococcus gordonii-Candida albicans Mono-Species and Dual-Species Biofilms.</a> Frontiers in Microbiology. 10, 2328 (2019).</li><li>Chaffin, W. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Candida+albicans+cell+wall+proteins.">Candida albicans cell wall proteins.</a> Microbiology and Molecular Biology Reviews. 72 (3), 495-544 (2008).</li></ol>

Small molecules from natural product sources (e.g., plants) include complex secondary metabolites that are highly diverse in chemical structure. They are believed to be involved in plant defense mechanisms. In addition, polypeptides are also present in plant tissues22. These natural product small molecules are rich sources of test molecules for drug discovery and development. However, the difficult and tedious methods required for their isolation and purification limit their use for therapeutic ap...

The purification of biomolecules from complex biological samples is an essential and often difficult step in biological experiments1. Isoelectric focusing (IEF) is well-suited for high resolution separation of complex biomolecules where carrier ampholytes travel according to their charge and establish the pH gradient in an electric field3. The first commercial carrier ampholyte for IEF was developed by Olof Vesterberg in 1964 and patented4,5. Carrier ampholytes are aliphatic oligo-amino oligo-carboxylic acid molecules of varying length and branching6. Subsequently, Vesterberg and others improved the carrier ampholytes for their expanded use in separating biomolecules6,7.
Methods to separate biomolecules include agarose and polyacrylamide gel electrophoresis, two-dimensional gel electrophoresis (2-DE), isoelectric focusing, capillary electrophoresis, isotachophoresis and other chromatographic techniques (e.g., TLC, FPLC, HPLC)2. Liquid-phase IEF performed in an instrument called a &#8220;Rotofor&#8221; was invented by Milan Bier8. He pioneered the concept and design of this instrument and contributed extensively to the theory of electrophoretic migration. His team also developed a mathematical model of electrophoretic separation process for computer simulations9.
The liquid-phase IEF apparatus is a horizontally rotating cylindrical cell consisting of a nylon core divided into 20 porous compartments and a circulating water cooling ceramic rod. The porous chambers allow molecules to migrate through the aqueous phase between the electrodes and permit collection of purified samples under vacuum in fractions. This purification system can provide up to 1000-fold purification of a specific molecule in &#60;4 hours. A valuable feature of this instrument is that it can be applied as a first step for purification from a complex mixture or as a final step to achieve purity10. If the molecule of interest is a protein, another advantage is that its native conformation will be maintained during the separation.
The use of liquid-phase IEF has been reported widely for proteins, enzymes and antibody purification6,10,11,12,13,14. Here we describe the use of this approach for separating and purifying small molecules and peptides from the medicinal plant Gymnema sylvestre. This protocol will help researchers concentrate and purify active small molecules from a plant extract for downstream applications at low cost. In addition, we also demonstrate that enrichment of proteins from a complex protein extract from Candida albicans fungus15 in this IEF-based system as a second example.

<table>
	<tbody>
		<tr>
			<td>0.45 &#181;m syringe filter</td>
			<td>Fisher scientfic</td>
			<td>09-720-004</td>
			<td></td>
		</tr>
		<tr>
			<td>2-Mercaptoethanol</td>
			<td>Sigma</td>
			<td>M3148</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium carbonate</td>
			<td>Sigma-Aldrich</td>
			<td>207861-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Bio-Lyte 3/10 Ampholyte</td>
			<td>Bio-Rad</td>
			<td>163-1113</td>
			<td></td>
		</tr>
		<tr>
			<td>Bio-Lyte 5/8 Ampholyte</td>
			<td>Bio-Rad</td>
			<td>163-1192</td>
			<td></td>
		</tr>
		<tr>
			<td>Compact low temperature thermostat</td>
			<td>Lauda -Brinkmann</td>
			<td>RM 6T</td>
			<td>Set water cooling to 4 oC and it can be run even at 0 oC as when it passes through the Rotofor cooling core, the circulating water temperature will be around 5 or more depending on the voltage.</td>
		</tr>
		<tr>
			<td>Coomassie Brilliant Blue R</td>
			<td>Sigma-Aldrich</td>
			<td>B7920</td>
			<td></td>
		</tr>
		<tr>
			<td>Dialysis tubing (3,500 MWCO)</td>
			<td>Spectrum Spectra/Por</td>
			<td>132112T</td>
			<td></td>
		</tr>
		<tr>
			<td>Gymnema plant leaf extract powder (&#62;25% Gymnemic acids)</td>
			<td>Suan Farma, NJ USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>Lab companion</td>
			<td>SI 300R</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>Leica</td>
			<td>DM 6B</td>
			<td></td>
		</tr>
		<tr>
			<td>Mini protean electrophoresis</td>
			<td>Bio-Rad</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>pH meter</td>
			<td>Mettler Toledo</td>
			<td>S20</td>
			<td>Useful to determine the pH of the Rotofor (liquid-phase IEF) fractions</td>
		</tr>
		<tr>
			<td>Rotofor</td>
			<td>Bio-Rad</td>
			<td>170-2972</td>
			<td>http://www.bio-rad.com/webroot/web/pdf/lsr/literature/M1702950E.pdf (Rotofor Instruction manual for assembling the unit)</td>
		</tr>
		<tr>
			<td>RPMI-1640 Medium</td>
			<td>HyClone</td>
			<td>DH30255.01</td>
			<td></td>
		</tr>
		<tr>
			<td>Sealing tape</td>
			<td>Bio-Rad</td>
			<td>170-2960</td>
			<td>Scotch tape may also be used.</td>
		</tr>
		<tr>
			<td>Sorvall legend micro 17 centrifuge</td>
			<td>Thermo scientific</td>
			<td>75002432</td>
			<td></td>
		</tr>
		<tr>
			<td>TPP tissue culture plate -96 well flat bottom</td>
			<td>TPP</td>
			<td>TP92696</td>
			<td></td>
		</tr>
	</tbody>
</table>

separation of bioactive small molecules, peptides from natural sources and proteins from microbes by preparative isoelectric focusing (ief) method

Natural products derived from plants and microbes are a rich source of bioactive molecules. Prior to their use, the active molecules from complex extracts must be purified for downstream applications. There are various chromatographic methods available for this purpose yet not all labs can afford high performance methods and isolation from complex biological samples can be difficult. Here we demonstrate that preparative liquid-phase isoelectric focusing (IEF) can separate molecules, including small molecules and peptides from complex plant extracts, based on their isoelectric points (pI). We have used the method for complex biological sample fractionation and characterization. As a proof of concept, we fractionated a Gymnema sylvestre plant extract, isolating a family of terpenoid saponin small molecules and a peptide. We also demonstrated effective microbial protein separation using the Candida albicans fungus as a model system.

Separation and purification of small molecules and peptides from Gymnema sylvestre plant extract 
Using the preparative liquid-phase IEF method, we fractionated medicinal plant extracts and cell surface proteins from a human pathogenic fungus, C. albicans. A schematic of these fractionation protocols is shown in Figure 1.
From 20 fractions of G. sylvestre extract obtained from liquid-phase IEF, the dark-colored mole...

Watch this Scientific Journal Video about Separation of Bioactive Small Molecules, Peptides from Natural Sources and Proteins from Microbes by Preparative Isoelectric Focusing IEF Method at JoVE.com

Separation of Bioactive Small Molecules, Peptides from Natural Sources and Proteins from Microbes by Preparative Isoelectric Focusing IEF Method

The objective is to fractionate and isolate bioactive small molecules, peptides from a complex plant extract, and proteins from pathogenic microbes by employing liquid-phase isoelectric focusing (IEF) method. Further, the separated molecules were identified and their bioactivity confirmed.

Separation of Bioactive Small Molecules, Peptides from Natural Sources and Proteins from Microbes by Preparative Isoelectric Focusing (IEF) Method

Natural products derived from plants and microbes are a rich source of bioactive molecules. Prior to their use, the active molecules from complex extracts ...

Our protocol describes the new findings on the separation and purification of active molecules from a plant extract. Scientists can use these methods to isolate small molecules and peptides from natural product sources for therapeutic users. Familiarize yourself with the instrument components and their assembly methods.It is important to be sure, that the sample to be separated is prepared by the suggested guidelines. This method involves multiple assembly processes of the instrument. And the visual demonstration will help users to follow and successfully apply them to the research.After assembling the liquid phase, IEF unit as described in the instruction manual, equilibrate the anion exchange membranes with 0.1 molar sodium hydroxide and the cation exchange membranes with 0.1 molar phosphoric acid. Align the rubber gaskets so that the three large oblong holes remain unblocked by the rubber. This will help exchange the ions between the electrode and the sample compartments via the membrane during electrophoresis.Assemble the inner and outer portion of the electrode by aligning the three oblong holes in the ion exchange gaskets. Then fill the electrodes with their respective electrolytes to prevent their membranes from drying out. Next, tighten the plastic screw holder to ensure a leak-free assembly.Cover the sample collection boards with sealing tape. Next assemble the parts of the focusing chamber over the ceramic cooling finger in the following sequence:anode electrode, nylon membrane core, focusing chamber and cathode electrode. Using a 50 milliliter syringe, fill the focusing chamber with pre-cooled distilled water.For a standard IEF cell, add 60 milliliters of water. Connect the IEF unit to circulating cooling water at four degrees Celsius. Operate the unit at 15 watts and 3000 volts until the voltage stabilizes, approximately three to five minutes.Then, switch off the power source. Using the fraction collector, remove the water from the cell. Re-seal the collection boards with sealing tape.Add 0.6 grams of Gymnema sylvestre plant extract, to 60 milliliters of distilled water. Dissolve the plant extract by mixing in a roller tube for five minutes. To remove the insoluble particles, centrifuge this solution at 10, 000 times G for five minutes.Transfer the supernatant to a 150 milliliter glass beaker and add ampholyte to create a 1%solution by volume, approximately 0.6 milliliters. Using a 50 milliliter syringe with a 1.5 inch 19 gauge blunt end needle, load this solution into the IEF cell through the sample collection boards. Then remove the cell from the stand and tap the electrode chamber, to dislodge and remove any bubbles.Connect the unit to the cooling water. With the power supply to constant 15 Watts begin fractionation. Run the IEF unit until the voltage reaches a constant value, approximately three hours.After pressing the harvest on button align the fraction collector pins with the collection boards. Push the pins through the sealing tape to begin collecting the Gymnema sylvestre fractions. Grow a single colony of C.albicans and yeast pepitone dextrose broth overnight at 30 degrees Celsius with shaking.Transfer the culture to a centrifuge tube and collect the yeast cells by centrifugation at 10, 000 times G for five minutes. After removing the supernatant, suspend the yeast cells in buffer and rotate the suspension in a roller tube for one hour at five degrees Celsius. To remove the yeast cells, centrifuge the suspension at 10, 000 times G for five minutes.Then filter the protein extract through a 0.45 micrometer filter. Transfer the protein extract to dialysis tubing and dialyze against water for 15 hours at four degrees Celsius. After estimating the protein concentration, collect a volume of protein extract containing 500 milligrams of total protein.Dilute the 500 milligrams of protein in 60 milliliters of water containing 1%ampholyte by volume. Using a syringe, load the diluted protein extract into the IEF cell and operate the unit at a constant 15 Watts for four hours. Since this instrument uses high voltage electric supply, the user must take all precautions to avoid any direct contact with the live electrodes.The fraction collector contains 20 plastic tubes, each 12 millimeters by 75 millimeters. And it's connected to the vacuum pump. Prepare to collect the fractions by pressing the harvest on button.Then align the 20 collection pins on the fraction collector with the 20 collection board so the sealed focusing cell. Push the collection pins through the sealing tape and simultaneously turn the vacuum pump on. Each tube will collect a protein fraction of about three milliliters.After reducing and boiling the protein fractions, analyze them on a 12.5%SDS page gel. Stain the gel with Coomassie Blue dye and place it on a rocker at room temperature. After two to three hours destain the gel.Then record the gel image using a gel imager. Prepare a yeast cell suspension by diluting an overnight culture of C.albicans at a 1:1000 ratio in RPMI cell culture medium supplemented with 50 millimolar glucose. Add 90 microliters of the cell suspension to each well of a 96 well plate.Next, add 10 microliters of each G.sylvestre fraction to separate wells. As a negative control, add 10 microliters of water with 1%ampholyte into separate wells. After incubating the 96 well plate at 37 degrees Celsius for 12 hours, observe the plate under a microscope.The lack of filamentous growth indicates the inhibition of C.albicans yeast-to-hypha conversion. Twenty fractions of G.sylvestre extract were obtained by liquid phase IEF. Dark colored molecules, the gymnemic acids, migrated to and were enriched at the anode end of the unit.Translucent light yellow fractions were observed at the cathode end. Aliquots from each G.sylvestre refraction were reduced, boiled and resolved by SDS page. A Coomassie Blue stain showed a different polypeptide band at about five kilodaltons, enriched in fractions 16 through 19.The Gymnemic acids in fraction one, and the next few fractions were not detected since they are not proteinaceous. However, these molecules can be separated by TLC and detected under UV light. Fraction 10 did not contain a detectable amount of these small molecules suggesting that most of the organic small molecules were enriched in fractions one through three.All 20 G.sylvestre fractions were assayed for inhibition of yeast-to-hypha conversion and hyphal growth in C.albicans. The greatest inhibition was observed in fraction one and little to no inhibition was observed in fractions 10 and above. Cell surface proteins from C.albicans were fractionated using liquid phase IEF.And aliquots from 18 fractions were analyzed by SDS page. Enriched proteins were visible in several fractions. The start, small molecules could not be separated by isoelectric focusing because they are believed to be non-ambulatory.Our studies shows they are ambulatory, at least weekly, and this method can be explored further for other small molecules.

separation-bioactive-small-molecules-peptides-from-natural-sources

Research

JoVE Journal

Immunology and Infection

9.3K vistas.  Kansas State University. Nuestro protocolo describe los nuevos hallazgos sobre la separación y purificación de moléculas activas de un extracto de planta. Los científicos pueden utilizar estos métodos para aislar moléculas pequeñas y péptidos de fuentes de productos naturales para los usuarios terapéuticos. Familiarícese con los componentes del instrumento y sus métodos de montaje.Es importante estar seguro de que la muestra que se va a separar está preparada siguiendo las directrices sugeridas. Es...

Video: Separation of Bioactive Small Molecules, Peptides from Natural Sources and Proteins from Microbes by Preparative Isoelectric Focusing IEF Method