![]() Mix the components well and place the solution on ice to cool. To prepare the organic phase of the emulsion, in a 50 milliliter tube weigh 0.2 grams of Tween 80, 1.8 grams of Span 80, and 38 grams of mineral oil. To prepare a chlorophyll a stock solution re-dissolve the dry chlorophyll a in 2-4 milliliters of 100%methanol. Then, using a rotary evaporator, evaporate the solvent until the chlorophyll a is completely dry. With thin-layer chromatography, verify chlorophyll a purity using a 68:25:5:2 volume-to-volume ratio of dichloromethane and hexane, isopropanol and methanol to elute the sample. You may use a flashlight to briefly illuminate the column during elution in order to observe the actual pigment colors. Then use a 3:1 volume-to-volume acetone-methanol mixture to elute the chlorophyll a. Using a Pasteur pipette gently load the sample onto a DEAE-Sepharose column equilibriated in 100%acetone, and with 100%acetone, elute the carotenoids. To proceed with chlorophyll a preparation partially dissolve the dry pigments in the smallest volume possible of 100%acetone, then add 4 milliliters of acetone to the suspension and swirl the flask to completely dissolve the pigments. If stopping at this point, keep the dry pigments under nitrogen or argon at 20 degrees Celsius in the dark until further processing. Next, using a rotary evaporator with a water bath that does not exceed 30 degrees Celsius evaporate the methanol until the extract is completely dry then use a small volume of diethyl ether to completely dissolve the pigments from the dried extracts and filter the solution through cotton wool, using additional small volume of ether to wash pigments off the cotton wool.Įvaporate the diethyl ether until the pigments are completely dry. When the color of the eluted fraction turns from dark green to pale green stop the elution. The actual color may vary from orange-green to green depending on the cyanobacteria culture and growth conditions.Įxchange the acetone with 100%methanol and collect the green fraction containing chlorophyll a. After washing, discard the eluted orange-green fraction. Load the crushed cells onto a glass column and with about 50-100 milliliters of 100%acetone wash the tissue to remove the carotenoids. The critical step during the chlorophyll extraction is to make sure that cyanobacteria cells are thoroughly lyophilized and to ensure the complete removal of water from the DEAE-Sepharose by several washes with acetone. To begin, weigh about five grams of lyophilized Spirulina platensis cells or other cyanobacterium cells containing only chlorophyll a or chlorophyll-a in thylakoid membranes and use a mortar and pestle to crush it. While preparing the following stock solutions perform all chlorophyll preparation steps under a chemical hood under green light or in the dark to minimize photo damage.Īlways add nitrogen or argon before freezing the pigments for storage and ensure that all solvents are analytical grade. Demonstrating the procedure will be Dominica Bednarczyk, a post-doc in my research group. Thus, it is well-suited for screening assays. The main advantage of this technique is that it does not require tagging or immobilizing the proteins. This method enables assembly of chlorophylls with recombinant proteins, which allows rigorous studies of chlorophyll-protein interactions and opens up new possibilities for constructing novel chlorophyll-protein complexes. The overall goal of this technique is to enable incorporation of water-insoluble chlorophylls into the binding sites of water-soluble proteins.
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