Immunology and Infection
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Separation of Bioactive Small Molecules, Peptides from Natural Sources and Proteins from Microbes by Preparative Isoelectric Focusing (IEF) Method
Chapters
Summary June 14th, 2020
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.
Transcript
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.
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