The overall goal of this protocol is to perform an easy and straightforward analysis of glucosinolates in plants and other biological samples. This method, to extract and analyze glucosinolates can be used to answer key questions in plant-insect ecology, plant pathology, plant breeding, and food sciences. The main advantage of this technique is that it's well-validated and broadly applicable to a wide range of biological samples.
Though this method is mainly used to quantify glucosinolates in plant materials, it can also be applied to soils or prepared foodstuffs. This extraction procedure can be performed in almost any lab because you mostly use standard lab equipment. Generally, individuals new to this method will perform the analyzes and inspection better when they read and observe the movie at least once.
To begin the procedure, mix 10 grams of G-25 cross-linked dextrin gel with 125 milliliters of ultra-pure water. Store the mixture in a capped bottle at 4 degrees Celsius. Then, dissolve 10, 000 units of type H-1 arylsulfatase in 30 milliliters of ultra-pure water.
Add to this 30 milliliters of absolute ethanol and stir the mixture well. Centrifuge the mixture at 2, 650 times G for 20 minutes at room temperature. Combine the supernatant with 90 milliliters of absolute ethanol in a beaker.
Mix and pour in new centrifuge tubes. Centrifuge the mixture at 1, 030 times G for 15 minutes at room temperature. Discard the supernatant.
Dissolve and combine the pellets in a total of 25 milliliters ultra-pure water. Vortex the mixture well and then transfer the mixture into 1 milliliter tubes. Store the sulfatate samples at 20 degrees Celsius for up to one year.
Next, weigh about 90 milligrams of sinogram and record the weight to 1 microgram accuracy. Then, dissolve the sinogram monohydrate in 10 milliliters of ultrapure water. Prepare from this sinogram stock solution five reference solutions with concentrations ranging from 50 to 750 micromolar.
Store the reference solutions in 1.5 milliliter tubes at 20 degrees Celsius. Next, for each sample and reference, label a 2 milliliter microcentrifuge tube in a column rack position. Pierce each microcentrifuge tube cap with a dissecting needle for later freeze-drying.
Place the labeled tubes in a block in the same configuration and spacing as the labeled columns. To prepare the glass pipette columns, use a wooden or glass rod to gently press a 1 centimeter by 1 centimeter piece of glass wool into the pipette. Pack the glass wool at the transition of the pipette barrel to stem.
Place a pipette column in each labeled position on the rack. Position the rack over a waste tray. Cut off the end of a plastic 1 milliliter pipette tip.
Shake the prepared dextrin gel well. And then use the widened pipette tip to load 0.5 milliliters of prepared dextrin gel into each column. Check the columns for leaking dextrin gel and replace any leaking columns.
Once all columns have been loaded with dextrin gel, wash each column with 1 milliliter of ultrapure water. Weigh between 50 and 100 milligrams of free stride, finely ground plant material in a 2 milliliter reaction tube with safety cap, and record the weight with 0.1 milligrams accuracy. Place two 3 millimeter diameter metal spheres in each tube.
Add to each tube 1 milliliter of 70%methanol in ultrapure water, and briefly vortex each mixture. Close the tubes with additional safety caps, and place them immediately in a 90 to 92 degrees Celsius water bath. Heat the tubes until the sawput begins to boil.
Transfer the tubes to an ultrasonic bath, and sonic heat the samples for 15 minutes. During sonication, begin thawing the sulfatate sample and sinogram references. After sonication, centrifuge the samples at 2, 700 times G for 10 minutes at room temperature.
Load the supernatants and thawed sinogram references onto the corresponding columns, being careful not to draw plant matter into the pipette. Add 1 milliliter of 70%methanol to each tube. Vortex the mixtures.
And ultrasonic heat the mixtures for 15 minutes. Centrifuge the tubes again under the same conditions as before. Load the supernatants onto the corresponding columns.
Add two 1 milliliter portions of 70%methanol to each column, allowing the columns to run dry between each portion. Wash residual methanol from each column with 1 milliliter of ultrapure water. Then, wash each column with two 1 milliliter portions of 20 millimolar sodium acetate buffer, pH 5.5.
Once the last portions of sodium acetate buffer have drained into the waste rack, remove the rack from the waste tray and dry the feet of the rack with tissue. Position the rack over the block of labeled microcentrifuge tubes, so the column tips are in the corresponding tubes. Load 20 microliters of sulfatate solution onto each column, ensuring that the solution reaches the surface of the column material.
Flush the sulfatate solution into the column material with 50 microliters of sodium acetate buffer. It's very important that the sulfatate is washed down into the column. The enzyme must be on the column to react to the intact glucosinolates bound on the column.
Once the sulfatate solution has been washed down onto each column, cover the columns with aluminum foil. Allow the columns to stand overnight. Next, allude the de-sulfo-glucosinolates from each column with two 0.75 milliliter portions of ultrapure water.
Once the columns have run dry, remove the column rack and cap each tube. Freeze the tubes in liquid nitrogen, or at 80 degrees Celsius for at least 30 minutes. Freeze dry the samples for 12 to 24 hours.
Dissolve each residue in 1 milliliter of ultrapure water. And then transfer each sample and reference to labeled HPLC sample vials. Separate the extracts on a reverse phase CAT column.
Use a detection wavelength of 229 nanometers. Once separated by HPLC, the glucosinolates can be identified by comparison of retention times and UV spectra with known glucosinolate standards. The glucosinolate concentrations in the sample are calculated from a sinogram calibration curve, and literature values for response factors.
From this, the glucosinolate concentrations in the original plant sample can be determined. Under the HPLC conditions used, the unhealthy progoitrin alludes quite early, and is separated from the biologically beneficial glucoraphanin. The endo-glucosinolates are also well separated.
Lyotropic series are observed with increasing side chain links for alconol, methylfol-alconol, and longer chain methyl-solfinol-glucosinolates. Unknown glucosinolates thus can be tentatively classified based on these lyotropic series in combination with UV spectra. With proper preparation, 150 to 200 samples can be extracted by one person in one working day.
It will take another day to allude to columns freeze-dry the samples, and prepare them for HPLC. Following this procedure, other methods, such as LCMS, can be applied to identify unknown desulfo-glucosinolates in your chromatogram. After watching this video, you should have a good understanding of how to extract glucosinolates from your biological samples and analyze them via HPLC.
Don't forget that working with methanol can be extremely hazardous, and should be always done under the fume hood.
