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 H3PO4) between runs and do not allow to dry out.</li>
	</ol>
	</li>
	<li>Assemble the inner and outer portion of the electrode by aligning three oblong holes in the ion-exchange gaskets. Fill the electrodes with respective electrolytes (~25-30 mL) to prevent their membranes from drying out.
	<ol>
		<li>Do not add excess (more than 1/3rd of electrode chamber volume) electrolyte that may build up pressure inside the electrode and cause leakage.</li>
	</ol>
	</li>
	<li>Cover the sample collection ports with sealing tape that comes with the instrument assembly parts (see Table of Materials). The sample collection ports side can be identified by the two vertical metal aligning pins. Alternatively, use standard sealing tape to seal the ports.</li>
	<li>Assemble all parts of the focusing chamber assembly in sequence (anode electrode, nylon membrane core, focusing chamber and cathode electrode) over the ceramic cooling finger.</li>
	<li>Fill the focusing chamber with precooled distilled water (total volume of 60 mL for the standard IEF cell) using a 50 mL syringe.</li>
	<li>Connect the liquid-phase IEF instrument to a circulating cooling water at 4 &#186;C and prerun the unit at 15 W and 3,000 V for 3-5 min or until the voltage stabilizes. 
	NOTE: Generally, within one minute, the voltage will reach the maximum set values. Prerunning with distilled water helps to remove the residual ions from the focusing chamber and the nylon membrane core.</li>
	<li>Switch off the power source and remove the water from the cell using the fraction collector. Re-seal the collection ports with sealing tape. 
	NOTE: Now the instrument is ready for use in step 2.4.</li>
</ol>
2. Separation and purification of small molecules and peptides from Gymnema sylvestre&#160; extract
<ol>
	<li>Measure 0.6 g of plant extract and dissolve in distilled water (60 mL) by mixing in a roller tube for 5 min. 
	NOTE: Any biological sample that is soluble and free of salt can be used for separation and purification using this IEF instrument. Samples with buffering salt concentration up to 10 mM can be used with slightly decreased resolution. We are interested in the saponin family of triterpenoid compounds, the gymnemic acids, from G. sylvestre plant for their unique antifungal properties16.</li>
	<li>Centrifuge the solubilized plant extract at 10,000 x g for 5 min to remove insoluble particles.</li>
	<li>Transfer the supernatant (~60 mL) to an 80 mL centrifuge tube, and mix it with 0.6 mL of ampholyte (pH 3-10) to 1% (v/v).</li>
	<li>Follow steps 1.1 &#8211; 1.7 to prepare the liquid-phase IEF unit. The chamber is now ready to load the sample.</li>
	<li>Use a 50 mL syringe with a 1-1/2 inch 19 G blunt end needle (comes with the instrument) and load the prepared sample with ampholyte (60 mL total) in the cell through sample collection ports.</li>
	<li>Remove air bubbles from the sample cell by removing the focusing cell from the stand and tapping the electrode chamber to dislodge the bubbles. The presence of air bubbles will cause voltage and current fluctuations and affect the run.</li>
	<li>Connect the unit to the water coolant (4 &#176;C) and start fractionation with the power supply at a constant 15 W.</li>
	<li>Run the apparatus for 3 h or until the voltage reaches a constant value. 
	NOTE: As the sample starts focusing, the voltage will start climbing gradually until it reaches a constant value.</li>
	<li>After the run, prepare to collect the fractions in the harvest box (containing 20 plastic tubes, 12 mm x 75 mm, 5-6 mL volume) connected to the vacuum pump by pressing the harvest button ON in the IEF unit.</li>
	<li>Align the 20-collection pins with the 20-collection ports of the focusing cell that is sealed with the tape.</li>
	<li>Push the collection pins through the sealing tape and turn the vacuum pump ON simultaneously (see Figure 2B, 2C &#38; 2D). 
	NOTE: About 3 mL fractions from each chamber will be collected into the tubes for each time. Fractions can be used for subsequent downstream applications (SDS-PAGE for peptide quantification, TLC and bioassay for small molecules).</li>
</ol>
3. Separation and purification of proteins from&#160;&#160;C. albicans
<ol>
	<li>Grow single colony of C. albicans in yeast-peptone-dextrose (YPD) broth16 at 30 &#176;C with shaking (200 rpm) for overnight.</li>
	<li>Collect the yeast cells by centrifugation (10,000 x g for 5 min).</li>
	<li>Suspend C. albicans yeast cells in ammonium carbonate (1.89 g/L) buffer containing 1% (v/v) beta-mercaptoethanol (&#946;-ME) (1/10th of the culture volume) and rotate in a roller tube for 1 h at 5 &#176;C15.</li>
	<li>Remove the yeast cells by centrifugation (10,000 x g for 5 min) and filter the protein extract through a 0.45 &#181;m filter. 
	NOTE: Protein samples from C. albicans cytosol, membrane or cell wall can be prepared and used for IEF fractionation. Similarly, proteins from bacteria or other biological samples (animal tissue extract) can be used after removing any salt by appropriate methods (e.g., by dialysis or using desalting columns).</li>
	<li>Dialyze the protein extract in a dialysis tubing (3,500 MWCO) against water for 15 h at 4 &#176;C. Estimate the protein concentration by the Bradford dye-binding method using gamma globulin as a standard17.</li>
	<li>Use 500 mg of total protein in 60 mL of water containing 1% (v/v) ampholyte (pH 5-8) for the fractionation. 
	NOTE: Since a broad-range ampholyte (pH 3-10) does not enrich certain non-glucan attached cell wall proteins of C. albicans well, use a narrow-range (pH 5-8) ampholyte. An ampholyte concentration of up to 2% can be used if the sample protein concentration is more than 2 mg/mL; this minimizes protein aggregation during focusing. Always keep the samples, ampholytes, and water precooled on ice.</li>
	<li>Repeat steps 1.1- 1.7 to prepare the IEF unit and use the protein solution from step 3.6 to load into the IEF cell.</li>
	<li>After 4 h of focusing at a constant 15 W, harvest protein fractions (1-20) as described above (steps 2.9-2.11) and analyze on 12.5% SDS-PAGE after reducing and boiling the protein samples18.</li>
	<li>Stain the resolved proteins by staining the SDS-PAGE gel with the Coomassie blue dye (0.01%) solution for 2-3 h at room temperature on a rocker. Destain the gel and record the gel image using a gel imager. 
	NOTE: Coomassie blue dye (0.01%) can be prepared by mixing 0.01 g of Coomassie blue powder in a destaining solution containing 40% methanol and 10% acetic acid.</li>
</ol>
4. Bioactivity of purified small molecules from plant extract Gymnema sylvestre
<ol>
	<li>Grow C. albicans in yeast cells as in step 3.1.</li>
	<li>To prepare cell suspension, dilute overnight culture of C. albicans yeast cells (1/1000 dilution) into a fresh RPMI cell culture medium supplemented with 50 mM glucose. 
	NOTE: C. albicans converts from yeast growth to hyphae under hyphal inducing conditions (RPMI at 37 &#176;C). Gymnemic acid small molecules are shown to inhibit the conversion of yeast cells into hyphae under hypha inducing conditions16. We aimed to determine if liquid-phase-IEF-separated G. sylvestre extract contain these bioactive molecules.</li>
	<li>From the prepared cell suspension, add 90 &#956;L to each well of the 96-well plate.</li>
	<li>From each fraction (1-20 harvested fractions of G. sylvestre extract obtained from liquid-phase IEF, Figure 4), add 10 &#956;L into the wells with 90 &#956;L of the above cell suspension (step 4.2). Perform the assay in triplicate.</li>
	<li>Add 10 &#956;L of water that contains ampholyte (1%) into separate wells with 90 &#956;L of C. albicans yeast cell suspension as a negative control. 
	NOTE: Ampholytes have bioactivity potential and so it is essential to include an ampholyte control while performing any bioassay19.</li>
	<li>Incubate the 96-well plate at 37 &#176;C for 12 h and observe the inhibition of C. albicans yeast-to-hypha conversion under the microscope16. Also, determine the percentage of inhibition of yeast-to-hypha conversion.</li>
</ol>

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<li>Yu, J. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Francisella+tularensis+T-cell+antigen+identification+using+humanized+HLA-DR4+transgenic+mice%5BTitle%5D)+AND+%22Clinical+Vaccine+Immunology%22%5BJournal%5D)">Francisella tularensis T-cell antigen identification using humanized HLA-DR4 transgenic mice.</a> Clinical Vaccine Immunology. 17 (2), 215-222 (2010).</li>
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The authors would like to declare no competing financial interests.

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 applications. The liquid-phase IEF approach used in this report highlights the ability to separate these small molecules and polypeptides without compromising their bioactivities.
This IEF based method offers several advantages in separating biological molecules including concentration of purified proteins from a complex mixture, maintenance of native conformation during and after their focusing, and collection of samples as individual purified fractions without cross-contamination. When necessary, samples can be re-focused with a narrow pH range to purify protein isoforms. Since a miniature IEF focusing cell (~15 mL) is available, it can be used for smaller volumes of samples as well. The new finding from this report is that organic small molecules and peptides can be separated from a complex plant extract. Though it is difficult to agree that small molecules can be separated from natural product extracts by IEF, it is plausible for those compounds that are amphoteric. The gymnemic acids that were separated from the gurmarin peptide in the G. sylvestre extract appear to be amphoteric as they contain a carboxylic acid group or they behave so at least in the presence of the ampholyte used. Since glycosides are bioactive natural molecules similar to gymnemic acids, the IEF method can be used to separate them from complex natural sources. Similarly, peptides from natural products may also be isolated using this liquid-phase IEF approach.
Some of the limitations in this approach are that not all small molecules can be fractionated by the IEF method as they must be water-soluble and weakly amphoteric, at least. The extract used here was prepared by a 50% methanol extraction of dried plant material but is water-soluble. The use of the IEF method for solvent-soluble and amphoteric compounds remains to be seen as some of the organic solvents are incompatible with the liquid-phase IEF instrument components. The tendency of proteins to precipitate at their isoelectric points (pI) in low ionic strength solutions is well known. However, in a rotating IEF system, protein precipitation is reduced as the focused proteins remain in circulation at their pI point.
If one uses a high concentration of proteins in this IEF separation, precipitation may occur. To minimize protein precipitation and to improve protein focusing, urea can be used up to 3-5 M. Nonionic detergents such as CHAPS, digitonin and low concentration of detergents (0.1-1%) can also be used to reduce protein aggregation during IEF. However, urea and detergents need to be removed before analyzing the proteins for their activity and in some cases, these agents may affect protein functions. A few critical steps to consider during a liquid-phase IEF run include loading the IEF focusing cell without air bubbles, replacing the ion exchange membranes if they were damaged, and replacing the vent buttons after a certain number of repeated uses.
In conclusion, using the liquid-phase IEF method, we have shown the separation of bioactive gymnemic acids and gurmarin polypeptide from the G. sylvestre leaf extract. Further, liquid-phase IEF can be useful to enrich selective proteins from the complex crude extracts of pathogenic microbes.

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 &amp;micro;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 (&gt;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 molecules (terpenoid saponins) were found to migrate and be enriched at the anode end (pH 2-3) and the light-yellow clear fractions were observed at the cathode end (pH 8-9) (Figure 2). Aliquots (20 &#956;L) from each fraction (1-20) were resolved on 15% SDS-PAGE after reducing and boiling the samples. A Coomassie blue-stained gel shows the diffused polypeptide band of about 5 kDa that is enriched in fractions 16-19 (Figure 3). It has been reported that the G. sylvestre plant contains a 35 amino acid gurmarin basic polypeptide with the predicted molecular weight of 4,209 Da20. Bacteria, plants and animals contain peptides; many of them are circular (knottins) and stable with wide range of biological activities such as insecticidal and antimicrobial properties21,22.
Biological activity of separated gymnemic acids 
The G. sylvestre plant also contains gymnemic acids (terpenoid saponins) as major constituents16,23,24. As expected, these small molecules in fraction 1 and the next few fractions were not detected by SDS-PAGE and Coomassie staining (Figure 3) since they are non-proteinaceous. However, these small molecules can be separated by TLC and detected under UV light (Figure 4A, lane F1). Fraction 10 did not contain a detectable amount of these small molecules on TLC suggesting most of the organic small molecules were enriched in fractions 1-3. Gymnemic acids (GAs) molecules were shown to inhibit C. albicans yeast-to-hypha transition16,25. We assayed all 20 fractions collected in this study for their inhibitory activity against C. albicans yeast-to-hypha conversion and hyphal growth16. The results are shown in Figure 4B,4C. The highest activity is observed in fraction 1, which agrees with the TLC results where several spots can be seen. Isomers of gymnemic acids exist and all have similar biological activities10. These isomers were separated in fraction 1-3 and show inhibition of C. albicans hyphal growth (Figure 4A,4C, fraction 1). The degree of hyphal inhibition was gradually decreased as it goes from 1 to 10. Little or no activity was obtained in fractions 10 and above.
Separation of cell surface proteins from pathogenic fungus, C. albicans 
Results from liquid-phase IEF fractionation of C. albicans cell surface proteins are shown in Figure 5. These cell surface proteins play important roles in C. albicans adhesion and pathogenesis26. Several enriched proteins (arrows) in different fractions were observed. This may allow identification of their immunological reactions with Candida infected human serum and/or their identification by mass spectrometry. Similarly, proteins from other cellular fractions (e.g., cytoplasm and cell wall) can be fractionated using this IEF method. The liquid-phase IEF-based purification will allow identification of low abundance proteins from complex biological samples, when coupled with mass spectrometry analysis.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61101/61101fig01.jpg" /> 
Figure 1: Flow chart showing the experimental workflow.&#160;Stepwise liquid-phase IEF fractionation procedures and subsequent downstream assays are depicted. Samples include Gymnema sylvestre leaf extract (sample 1) and Candida albicans non-glucan attached yeast proteins (sample 2). <a href="https://www.jove.com/files/ftp_upload/61101/61101fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61101/61101fig02.jpg" /> 
Figure 2: Liquid-phase IEF apparatus setup and fractionation of G. sylvestre plant extract.&#160;(A) During the run, (B) during fraction collection, (C) after fraction collection, and (D) liquid-phase IEF apparatus parts, 1) ion exchange membranes, 2) focusing chamber and membrane core, 3) electrode assembly (negative), 4) electrode assembly (positive). <a href="https://www.jove.com/files/ftp_upload/61101/61101fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61101/61101fig03.jpg" /> 
Figure 3: SDS-PAGE separations of the IEF focused plant extract fractions.&#160;L- ladder, PC- Positive control (peptide), 0 - input sample, 1-20 separated fractions. SDS-PAGE (15% resolving gel) was stained by Coomassie blue dye to visualize the resolved peptides (~5 kDa) from fractions 1-20. Fractions 1-3 contain small molecules (fraction 1 has darker color indicating enriched compounds) which cannot be stained/detected by Coomassie blue dye. <a href="https://www.jove.com/files/ftp_upload/61101/61101fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/61101/61101fig04.jpg" /> 
Figure 4: Analysis of IEF fractionated small molecules by TLC and determination of bioactivity against C. albicans.&#160;(A) Shows TLC analysis of small molecules from fraction #1 and #10. Activated silica gel plate was used to spot ~5 &#181;L of samples and ran with toluene: chloroform: methanol solvent (5:8:3 ratio) until the solvent front reached the margin. TLC separated compounds were detected under an epifluorescence UV light (310 nm). (B) Shows the % inhibition of C. albicans yeast-to-hypha conversion by different fractions. (C) Demonstrates the cell morphology of C. albicans under hypha inducing conditions. Fraction #1 shows maximum (98%) inhibition of yeast-to-hypha conversion. Other fractions and controls show no inhibition of C. albicans hyphal growth after 12 hours of incubation at 37 &#176;C. <a href="https://www.jove.com/files/ftp_upload/61101/61101fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/61101/61101fig05.jpg" /> 
Figure 5: SDS-PAGE analysis of C. albicans cell surface proteins (non-glucan attached).&#160;L- ladder, 0 - input sample, 1-18 fractions collected after IEF in a standard liquid-phase IEF cell using a narrow range (pH 5-8) ampholyte. The image shows SDS-PAGE (12.5%) resolved proteins after staining with Coomassie blue dye. Several proteins were enriched in certain fractions (arrows). <a href="https://www.jove.com/files/ftp_upload/61101/61101fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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 ...

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Nanyang Technological University

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JoVE Journal

Immunology and Infection

9.4K Views.  Kansas State University. 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.

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

Resolving Affinity Purified Protein Complexes by Blue Native PAGE and Protein Correlation Profiling

Most proteins act in association with others; hence, it is crucial to characterize these functional units in order to fully understand biological processes. Affinity purification coupled to mass spectrometry (AP-MS) has become the method of choice for identifying protein-protein interactions. However, conventional AP-MS studies provide information on protein interactions, but the organizational information is lost. To address this issue, we developed a strategy to unravel the distinct functional assemblies a protein might be involved in, by resolving affinity-purified protein complexes prior to their characterization by mass spectrometry. Protein complexes isolated through affinity purification of a bait protein using an epitope tag and competitive elution are separated through blue native electrophoresis. Comparison of protein migration profiles through correlation profiling using quantitative mass spectrometry allows assignment of interacting proteins to distinct molecular entities. This method is able to resolve protein complexes of close molecular weights that might not be resolved by traditional chromatographic techniques such as gel filtration. With little more work than conventional AP-geLC-MS/MS, we demonstrate this strategy may in many cases be adequate for obtaining protein complex topological information concomitantly to identifying protein interactions.

Here we present protocols for affinity purification of protein complexes and their separation by blue native PAGE, followed by protein correlation profiling using label free quantitative mass spectrometry. This method is useful to resolve interactomes into distinct protein complexes.

Most proteins act in association with others; hence, it is crucial to characterize these functional units in order to fully understand biological ...

Biochemistry

Large-scale Top-down Proteomics Using Capillary Zone Electrophoresis Tandem Mass Spectrometry

Capillary zone electrophoresis-electrospray ionization-tandem mass spectrometry (CZE-ESI-MS/MS) has been recognized as a useful tool for top-down proteomics that aims to characterize proteoforms in complex proteomes. However, the application of CZE-MS/MS for large-scale top-down proteomics has been impeded by the low sample-loading capacity and narrow separation window of CZE. Here, a protocol is described using CZE-MS/MS with a microliter-scale sample-loading volume and a 90-min separation window for large-scale top-down proteomics. The CZE-MS/MS platform is based on a linear polyacrylamide (LPA)-coated separation capillary with extremely low electroosmotic flow, a dynamic pH-junction-based online sample concentration method with a high efficiency for protein stacking, an electro-kinetically pumped sheath flow CE-MS interface with extremely high sensitivity, and an ion trap mass spectrometer with high mass resolution and scan speed. The platform can be used for the high-resolution characterization of simple intact protein samples and the large-scale characterization of proteoforms in various complex proteomes. As an example, a highly efficient separation of a standard protein mixture and a highly sensitive detection of many impurities using the platform is demonstrated. As another example, this platform can produce over 500 proteoform and 190 protein identifications from an Escherichia coli proteome in a single CZE-MS/MS run.

A detailed protocol is described for the separation, identification, and characterization of proteoforms in protein samples using capillary zone electrophoresis-electrospray ionization-tandem mass spectrometry (CZE-ESI-MS/MS). The protocol can be used for the high-resolution characterization of proteoforms in simple protein samples and the large-scale identification of proteoforms in complex proteome samples.

Capillary zone electrophoresis-electrospray ionization-tandem mass spectrometry (CZE-ESI-MS/MS) has been recognized as a useful tool for top-down ...

Chemistry

Cell-Lineage Guided Mass Spectrometry Proteomics in the Developing (Frog) Embryo

Characterization of molecular events as cells give rise to tissues and organs raises a potential to better understand normal development and design efficient remedies for diseases. Technologies enabling accurate identification and quantification of diverse types and large numbers of proteins would provide still missing information on molecular mechanisms orchestrating tissue and organism development in space and time. Here, we present a mass spectrometry-based protocol that enables the measurement of thousands of proteins in identified cell lineages in Xenopus laevis (frog) embryos. The approach builds on reproducible cell-fate maps and established methods to identify, fluorescently label, track, and sample cells and their progeny (clones) from this model of vertebrate development. After collecting cellular contents using microsampling or isolating cells by dissection or fluorescence-activated cell sorting, proteins are extracted and processed for bottom-up proteomic analysis. Liquid chromatography and capillary electrophoresis are used to provide scalable separation for protein detection and quantification with high-resolution mass spectrometry (HRMS). Representative examples are provided for the proteomic characterization of neural-tissue fated cells. Cell-lineage-guided HRMS proteomics is adaptable to different tissues and organisms. It is sufficiently sensitive, specific, and quantitative to peer into the spatio-temporal dynamics of the proteome during vertebrate development.

Cell-Lineage Guided Mass Spectrometry Proteomics in the Developing Frog Embryo

Here we describe a mass spectrometry-based proteomic characterization of cell lineages with known tissue fates in the vertebrate Xenopus laevis embryo.

Characterization of molecular events as cells give rise to tissues and organs raises a potential to better understand normal development and design ...

Isolating Small Extracellular Vesicles and Extracting Their Peptidome

Identification of Peptides of Small Extracellular Vesicles from Bone Marrow-Derived Macrophages

Small extracellular vesicles (sEVs) are typically secreted by the exocytosis of multivesicular bodies (MVBs). These nanovesicles with a diameter of &lt;200 nm are present in various body fluids. These sEVs regulate various biological processes such as gene transcription and translation, cell proliferation and survival, immunity and inflammation through their cargos, such as proteins, DNA, RNA, and metabolites. Currently, various techniques have been developed for sEVs isolation. Among them, the ultracentrifugation-based method is considered the gold standard and is widely used for sEVs isolation. The peptides are naturally biomacromolecules with less than 50 amino acids in length. These peptides participate in a variety of biological processes with biological activity, such as hormones, neurotransmitters, and cell growth factors. The peptidome is intended to systematically analyze endogenous peptides in specific biological samples by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Here, we introduced a protocol to isolate sEVs by differential ultracentrifugation and extracted peptidome for identification by LC-MS/MS. This method identified hundreds of sEVs-derived peptides from bone marrow-derived macrophages.

Author Spotlight: Peptidome Extraction from Small Extracellular Vesicles Isolated from Bone Marrow-Derived Macrophages 

This protocol describes a procedure to isolate small extracellular vesicles from macrophages by differential ultracentrifugation and extract the peptidome for identification by mass spectrometry.

Small extracellular vesicles (sEVs) are typically secreted by the exocytosis of multivesicular bodies (MVBs). These nanovesicles with a diameter of ...

Enhancing EV Isolation for Precise Biofluid Analysis

Chemical Affinity-Based Isolation of Extracellular Vesicles from Biofluids for Proteomics and Phosphoproteomics Analysis

Extracellular vesicles (EVs) from biofluids have recently gained significant attention in the field of liquid biopsy. Released by almost every type of cell, they provide a real-time snapshot of host cells and contain a wealth of molecular information, including proteins, in particular those with post-translational modifications (PTMs) such as phosphorylation, as the main player of cellular functions and disease onset and progression. However, the isolation of EVs from biofluids remains challenging due to low yields and impurities from current EV isolation methods, making the downstream analysis of EV cargo, such as EV phosphoproteins, difficult. Here, we describe a rapid and effective EV isolation method based on functionalized magnetic beads for EV isolation from biofluids such as human urine and downstream proteomics and phosphoproteomics analysis following EV isolation. The protocol enabled a high recovery yield of urinary EVs and sensitive profiles of EV proteome and phosphoproteome. Furthermore, the versatility of this protocol and relevant technical considerations are also addressed here.

Author Spotlight: Advancing EVtrap for High-Throughput Proteomics in Disease Biomarker Discovery 

The present protocol provides detailed descriptions for the efficient isolation of urinary extracellular vesicles utilizing functionalized magnetic beads. Moreover, it encompasses subsequent analyses, including western blotting, proteomics, and phosphoproteomics.

Extracellular vesicles (EVs) from biofluids have recently gained significant attention in the field of liquid biopsy. Released by almost every type of ...

Separating Protein with SDS-PAGE

Sodium Dodecyl Sulfate Poly-Acrylamide Gel Electrophoresis, or SDS-PAGE, is a widely-used technique for separating mixtures of proteins based on their size and nothing else. SDS, an anionic detergent, is used to produce an even charge across the length of proteins that have been linearized. By first loading them into a gel made of polyacrylamide and then applying an electric field to the gel, SDS-coated proteins are then separated. The electric field acts as the driving force, drawing the SDS coated proteins towards the anode with larger proteins moving more slowly than small proteins. In order to identify proteins by size, protein standards of a known size are loaded along with samples and run under the same conditions.
This video presents an introduction to SDS-PAGE by first explaining the theory behind it and later demonstrating its step-by-step procedure. Various experimental parameters, such as the polyacrylamide concentration and voltage applied to the gel are discussed. Downstream staining methods like Coomassie and silver stains are introduced, and variations of the method, like 2D gel electrophoresis are presented.

Separating Protein with SDS-PAGE: Procedure and Applications

Sodium Dodecyl Sulfate Poly-Acrylamide Gel Electrophoresis, or SDS-PAGE, is a widely-used technique for separating mixtures of proteins based on their ...

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Basic Biology

Basic Methods in Cellular and Molecular Biology

Chromatography-based Biomolecule Purification Methods

In biochemistry, chromatography-based purification methods are employed to isolate compounds from a complex mixture. Two such methods used commonly by biochemists are size-exclusion chromatography and affinity chromatography. In size-exclusion chromatography, a column packed with porous beads separates components of a mixture based on size. On the other hand, affinity chromatography allows for a more specific separation of biomolecules by using a column that is composed of stationary phase, which contains target-specific ligands.
This video serves as an introduction to size-exclusion and affinity chromatography, as well as the concepts that govern them. A step-by-step procedure for the purification of a histidine-tagged protein by immobilized metal affinity chromatography is described. Applications for both of these chromatographic methods in biochemistry and biomedical research are also profiled.
<div class="chapter_text" id="script_0">
&#34;Chromatography&#34; refers to a wide range of methods used to isolate a component from a complex mixture, an essential step before a biomolecule&#39;s properties and activities can be determined. Each chromatographic technique has a different mechanism for separation, depending on the sample matrix and target compound. This video will focus on the principles and operation of two methods common to biochemistry: size-exclusion and affinity chromatography.
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<div class="chapter_text" id="script_1">
Size-exclusion chromatography or SEC is based on the size of the compounds in the sample. A mobile phase containing the sample is added to a column with a porous material: the stationary phase. The molecules in the sample fall into 1 of 3 categories.
Molecules too large to enter the pores travel the shortest distance through the column. Any species with a molecular weight above this &#34;exclusion limit&#34; will exit the column at the same time. Molecules small enough to freely enter the pores will be retained the longest, and will exit the column together. The molecular weight allowing complete pore entry is the &#34;permeation limit&#34;.
Only molecules between these limits will be separated from one another, as they spend varying amounts of time diffusing into and out of the pores. Smaller molecules are retained longer on the column because they spend more time in the stationary phase, whereas larger molecules within these limits exit earlier.
Molecular weights across 1 to 2 orders of magnitude fall within these limits. Columns are chosen with this in mind, or multiple columns can be used in series if there is a wide range of desired compounds.
Now that you&#39;ve seen the theory of SEC, let&#39;s look at how it is carried out.
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<div class="chapter_text" id="script_2">
To begin the SEC procedure, the column must be equilibrated with deionized water and chromatography buffer. Once prepared, buffer containing the sample is injected onto the column. The buffer is then pushed through at a low flow rate. A detector monitors what exits the column to determine the presence of the desired analyte. Large molecules with molecular weights above the exclusion limit exit the column at the same time. Small fractions from the column are collected in tubes. Each fraction is tested for the quality of the target molecule by gel electrophoresis or other analytical techniques.
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<div class="chapter_text" id="script_3">
Now let&#39;s have a look at affinity chromatography or AC, one of the most efficient ways to purify proteins. Many biomolecules bind selectively to certain ligands-a property which AC utilizes by adhering a target-specific ligand to the stationary phase.
When the mixture flows through the column, the target molecules attach to the ligand, and the rest flow through. After the mixture has passed through the column, the target molecule can be collected through one of two elution methods based on specificity.
Biospecific elution can be said to a have a &#34;normal-&#34; or &#34;reverse-role&#34;. In normal-role biospecific elution, an agent is added that competes with the adhered ligand to bind with the target biomolecule.
In reverse-role biospecific elution, an agent competes with the target to bind to the adhered ligand. The second elution type, nonspecific elution, lowers the target-to-ligand binding by changing the solution&#39;s pH, ionic strength, or polarity. If a protein does not bind to a ligand that can be immobilized, the protein can be expressed containing a &#34;tag&#34;: short peptide sequences engineered to bind to the ligand.
One variety is immobilized metal ion affinity chromatography, &#34;IMAC&#34; for short, where an adhered metal ligand, like nickel or cobalt, binds to histidine residues on the modified protein. Through molecular biology techniques, target proteins are generated with repeating histidine residues, called a polyhistidine-tag, which binds to the metal via the imidazole side chain on histidine. Once bound, the protein can be collected with free imidazole via reverse-role specific elution and later used in a wide variety of downstream applications.&#160;Now that you&#39;ve seen the theory of affinity chromatography, let&#39;s look at an IMAC procedure in the laboratory.

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<div class="chapter_text" id="script_4">
In IMAC, the stationary phase can be added directly to the mixture of mobile phase and sample, allowing the binding of the his-tagged protein. This slurry is then poured into the column, where the non-bound compounds drip into waste, while the slurry remains. The slurry container is rinsed to collect residual resin and sample, which is added to the column.
The resin is stirred to ensure the unbound components are free-flowing. Added wash buffer helps flush them away. Once all of the unbound components have been removed, the waste is replaced with a container to collect the target protein.
Buffer containing imidazole is added, stirred, and allowed to rest to unbind the target molecule. The imidazole binds to the metal, replacing and releasing the tagged protein. The freed protein is collected, and the imidazole step is repeated to ensure total collection. To further purify the sample, SEC can be run on the sample prior to analysis.
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<div class="chapter_text" id="script_5">
Now that we&#39;ve seen the theory and procedure of these two techniques, let&#39;s look at some of the ways they&#39;re applied in the biochemical field.
A common reason to purify proteins is to study their role in disease. Cystic fibrosis is caused by defects in the cystic fibrosis transmembrane conductance regulator protein, or CFTR. After growing the protein with a his-tag in yeast, both affinity and size-exclusion chromatography allow the isolation of the protein, followed by the study of its function.
In some instances, the presence of a polyhistidine tag can change a protein&#39;s structure, thereby affecting its function. Another common tag is maltose-binding protein or MBP, which will bind to bound amylose in a column. Maltose is then used to release the complex. The MBP can then be cleaved and removed with SEC to produce the pure desired protein.
</div>
<div class="chapter_text" id="script_6">
You&#39;ve just watched JoVE&#39;s video on size-exclusion and affinity chromatography. It covered the theory of the techniques, went over general procedures, and covered some of the uses of the techniques.
Thanks for watching!
</div>

Size Exclusion and Affinity Chromatography

In biochemistry, chromatography-based purification methods are employed to isolate compounds from a complex mixture. Two such methods used commonly by ...

Two-Dimensional Gel Electrophoresis

Two-dimensional gel electrophoresis (2DGE) is a technique that can resolve thousands of biomolecules from a mixture. This technique involves two distinct separation methods that have been coupled together: isoelectric focusing (IEF) and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). This physically separates compounds across two axes of a gel by their isoelectric points (an electrochemical property) and their molecular weights.
The procedure in this video covers the main concepts of 2DGE and a general procedure for characterizing the composition of a complex protein solution. Three examples of this technique are shown in the applications section, including biomarker detection for disease initiation and progress, monitoring treatment in patients, and the study of proteins following posttranslational modification (PTM).
<div class="chapter_text" id="script_0">
Two-dimensional, or 2D, gel electrophoresis is a technique utilizing two distinct separation methods which can separate thousands of proteins from a single mixture. One of the techniques, SDS-PAGE or sodium dodecyl sulfate polyacrylamide gel electrophoresis, cannot fully separate complex mixtures alone. 2D gel electrophoresis couples the SDS-PAGE to a second method, isoelectric focusing or IEF, which separates based on isoelectric points, allowing for the resolution of potentially all proteins in a cell lysate. This video will show the principles of 2D gel electrophoresis, a general procedure, and some of its biomedical applications.
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<div class="chapter_text" id="script_1">
2D gel electrophoresis begins with IEF as the first dimension. Every protein has a pH value, called the isoelectric point or pI, where the net charge is zero. When a protein is subjected to an electric field, it will move toward the electrode with opposite charge. Samples of interest are loaded onto immobilized pH gradient, or IPG, strips which have embedded ampholytes, molecules containing both acidic and basic groups. An electric field is then applied to the pH gradient strip, causing the proteins to migrate until they reach the pH value matching their pI, where they lose their net charge.
Prior to running the second-dimension, the embedded proteins are treated with SDS, denaturing them and providing a uniform negative charge. Once completed, the IPG strips are placed onto a polyacrylamide gel. An applied electric field draws the proteins toward the anode, with larger proteins moving more slowly through the gel.
Once the protein mixture has been separated according to pI and molecular weight, the proteome map is visualized using stains, and proteins of interest are identified.
Now that we have discussed the principles of 2D gel electrophoresis, let&#39;s go over a typical laboratory procedure.
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<div class="chapter_text" id="script_2">
Before the experiment can be performed, the proteins must be solubilized into media. Solubilization of the sample is achieved by de-aggregating proteins with a combination of chaotropic agents for disrupting hydrogen-bonding interactions, nonionic detergents to prevent altering of the proteins&#39; charge, reducing agents to break disulfide bonds, and buffers. To remove interfering abundant proteins and other molecules, the material is sequentially extracted by centrifugation, and collection of the resulting pellet; followed by treatment with endonuclease, an enzyme used to consume any DNA that would interfere with the experiment.
Once the proteins have been solubilized, IPG strips are prepared by rinsing with a strip-cleaning solution and left upside-down to dry. Each strip is then assigned a strip holder number. Once ready, the cellular extract is loaded onto the strips in a slow, sliding motion from the negative to the positive end. For the purpose of rehydration, damp blotting paper is placed on top of the electrode and under the gel strips; the IPG strips are then lined onto the IEF instrument. A high electric current is applied, and the proteins begin to migrate.
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<div class="chapter_text" id="script_3">
Following completion of the first dimension, the gel for SDS-PAGE is prepared in a casting apparatus. The IPG strips are treated by placing them face down in SDS-containing equilibration buffer. The electrophoresis unit is readied with the addition of electrophoresis buffer. The treated IPG strips are collected using tweezers, placed on top of the gel plates, and sealed with agarose-sealing solution. A voltage source then applies an electric field, which is held until the fastest-moving proteins are 1 cm from the bottom of the gel.
After completion of the electrophoresis, the proteins must be visualized. Traditionally this is performed by staining with Coomassie blue or silver nitrate. Proteins of interest may be transferred from the gel, and analyzed by Western blot analysis.
A second identification approach involves excision of the proteins from the gel, digesting them, than analyzing them by mass spectrometry.
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Now that we&#39;ve reviewed a procedure, let&#39;s look at some of the uses for 2D gel electrophoresis.
One of the most common uses for this technique is the identification of molecules involved in disease initiation and progression. 2D gel electrophoresis, coupled with mass spectrometry, can detect the up- or down-regulation of specific proteins in diseased areas in comparison to healthy ones.
Additionally, 2D gel electrophoresis is useful in following the progress of patients&#39; response to a potential therapeutic drug. Specimens may be taken from patients at various timepoints following administration of the treatment. In this way, 2D gel electrophoresis coupled with Western blot or mass spectrometry analysis, can detect proteins associated with negative responses such as inflammation; or the absence of proteins in an alleviated state.
Another use for 2D gel electrophoresis is in the study of protein structure and function following posttranslational modification, or PTM, which are additions to proteins following their translation from mRNA. PTM&#39;s can regulate a variety of functions, including protein signaling, gene expression, or cause oxidative damage. 2D gel electrophoresis is sensitive to modifications such as methylation or acetylation, which can cause a shift in pI as well as the molecular weight.
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You&#39;ve just watched JoVE&#39;s video on 2D gel electrophoresis. This video described the principles of the technique, a typical experimental procedure, and several of its applications in the field of biomedicine.
Thanks for watching!
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Two-dimensional gel electrophoresis (2DGE) is a technique that can resolve thousands of biomolecules from a mixture. This technique involves two distinct ...

Peptide Purification: An RP-HPLC-based Technique to Extract Peptides from Digested Protein Lysates

Source: Cheng, L. C. et al. <a href="https://www.jove.com/video/57996" target="_blank">Phosphopeptide Enrichment Coupled with Label-free Quantitative Mass Spectrometry to Investigate the Phosphoproteome in Prostate Cancer.</a> J. Vis. Exp. (2018)
In this video, we demonstrate reversed-phase high-performance liquid chromatography to purify peptides from a digested protein lysate mixture. These purified peptides are then lyophilized and stored for downstream applications. Analysis of peptides can help understand the mechanics involved in cellular signaling and cancer.

Source: Cheng, L. C. et al. Phosphopeptide Enrichment Coupled with Label-free Quantitative Mass Spectrometry to Investigate the Phosphoproteome in ...

JoVE Encyclopedia of Experiments

Cancer Research

Prostate Cancer

Assays and techniques

Anion-Exchange Chromatography-Based Protein Purification: A Separation Technique to Isolate a Protein of Interest From Dialyzed Bacterial Lysate Based on Net Charge

Source: Fuehner, S., et al. <a href="https://www.jove.com/v/60065">Purification of Human S100A12 and Its Ion-induced Oligomers for Immune Cell Stimulation</a> J. Vis. Exp. (2019)
In this video, we demonstrate an anion-exchange chromatography technique to purify human calcium-binding protein S100A12 based on the net surface charge of the protein from an Escherichia coli culture.

Source: Fuehner, S., et al. Purification of Human S100A12 and Its Ion-induced Oligomers for Immune Cell Stimulation J. Vis. Exp. (2019)
In this video, we ...