To observe subcellular localization of GFP, after 48 hours of agroinfiltration of Nicotiana benthamiana, select a flat leaf and make a six-millimeter hole using a hole punch. Drip 30%sucrose solution onto a glass slide. Place the leaf sample with the back facing upwards, and cover it with a cover glass.
Collect the intact, fresh N.benthamiana leaves expressing recombinant GFP at three days post-agroinfiltration, and weigh the leaves. Rinse the leaf blade with pre-cooled, sterilized, double-distilled water to remove debris. Submerge 20 grams with the abaxial side down in 500 milliliters of pre-cooled 100-millimolar phosphate buffer in a 1, 000 milliliter wide-mouth beaker.
Cover the leaves with 100 mesh nylon, fine mesh yarn and a plastic plate. Connect the beaker to a vacuum pump. Apply 0.8 megapascal vacuum for one minute, then quickly restore to atmospheric pressure.
Remove leaves from the buffer and gently blot dry with absorbent paper. Roll up the leaves and wrap them with mesh yarn. For collecting apoplastic washing fluid, or AWF, position rolled leaves tip-side up in 50-milliliter centrifuge tubes with mesh yarn fastened by the tube caps.
Centrifuge the leaves at 500g for five minutes at four degrees Celsius. Remove the leaves and collect AWF using a pipette. Add Nickel Sepharose excel resin to a tube in a one-to-1, 000 ratio relative to the protein extract volume.
Wash nickel resin three times with 10 times the resin volume of double-distilled water and phosphate buffer separately, in turn. Incubate protein extract with nickel resin for two hours to allow full binding. Then, centrifuge at 500g for five minutes at four degrees Celsius to remove unbound proteins in the supernatant.
Wash three times with 20 times the resin volume of phosphate buffer. After the last wash, add 10 times the resin volume of 250 millimolar imidazole to elute GFPs. Centrifuge the mixture at 500g for five minutes at four degrees Celsius, and collect the supernatant.
Western blot analysis confirmed the transient expression of 30 kilodalton recombinant protein GFPs in N.benthamiana. Fluorescence microscopy illustrated the presence of GFPs within the apoplast. AWF proteins extracted by vacuum infiltration centrifugation also contained GFPs.
Out of the eight extraction solutions tested, phosphate buffer extracted the greatest amount of AWF and GFPs. Comparing phosphate buffer at varying pH revealed improved AWF and GFPs extraction at neutral to mildly alkaline conditions. Additionally, 100 millimolar phosphate buffer showed maximal efficacy for recovering AWF and GFPs.
With a 100-millimolar phosphate buffer, 495 micrograms of total AWF proteins per gram of fresh leaf were recovered from the apoplast. The extracted AWF contained 10 micrograms of GFPs, which corresponded to approximately 18%of the GFPs in the total soluble protein. Following nickel affinity chromatography, GFPs'purity reached 84.3%from AWF extraction, substantially higher than the 44.9%purity achieved through total soluble protein extraction.
