Isolate physiologically active thylakoid membranes

Friday, November 21, 2003

Description
Thylakoid membranes are prepared

Procedure
Thylakoid membranes are prepared by homogenizing 40 g of fresh, deveined, market spinach (Spinacia oleracea) in 200 mL of Grinding Buffer with a chilled kitchen blender at 5 degrees C. The spinach leaves are torn into small pieces not more than 4 cm per side and placed in the blender. The Grinding Buffer is poured on top of the leaves. The blender is pulsed at low speed several times for 1-3 seconds such that all of the large leaf fragments are brought down into the slurry. Once all of the leaf fragments are in the slurry, the slurry is homogenized at the highest possible speed for 20 seconds. The resulting homogenate is filtered through 8 layers of cheesecloth into a 400 mL beaker. About 35 mL of the dark green filtrate is placed in one 40 mL centrifuge tube and is centrifuged at 2500 X G for 4 minutes at 4 degrees C. The supernatant solution can be discarded by pouring into the sink. The dark green pellet containing many subcellular organelles (intact chloroplasts, thylakoid membranes, nuclei, mitochondria, etc) should be gently but thoroughly stirred with a small paint brush before adding 20 mL of Washing Buffer followed by some additional stirring with the paint brush. You are trying to get a uniform suspension of green material without significant numbers of "clumps." All subsequent suspensions should be done with the paint brush in the same way such that the suspensions are smooth and homogeneous in appearance. The resulting dark green suspension is centrifuged at 1000 X G for 20 seconds to remove the crude cell debris and more dense organelles such as nuclei. The supernatant fluid is carefully decanted into a clean 40 mL centrifuge tube and the pellet is discarded. The supernatant fluid is centrifuged at 5000 X G for 10 minutes. The resulting pellet is resuspended into 40 mL of Washing Buffer and centrifuged again at 5000 X G for 10 minutes. This final pellet is resuspended into 5 mL of Washing Buffer.

The chlorophyll concentration in the thylakoid suspension is determined by adding 0.10 mL of the suspension to 10 mL of 80% acetone in a 15 mL test tube. This solution is mixed by inverting 3 times and then filtered through a Whatman #4 filter paper into a large cuvette (10 X 105 mm) using a 50 mL glass funnel. The absorbance of the green solution is measured at 663 nm and at 645 nm using 80% acetone to zero the spectrophotometer. The concentration of chlorophyll in the original sample is calculated using the equation:

mg chl/mL = [(A663)(0.00802) + (A645)(0.0202)] X dilution factor
(in this case the dilution faction is 10 mL/0.1mL = 100)

Once the chlorophyll concentration is determined, the total chlorophyll yeild should be determined by multiplying the chlorophyll concentration in mg chl/mL times the volume (mL) of the thylakoid suspension. Once the chlorophyll concentration and the total chlorophyll yeild is known, the chlorophyll concentration should be adjusted to about (it doesn't have to be exact) 0.5 mg chl/mL by adding the appropriate amount of Washing Buffer to existing thylakoid suspension or by centrifuging again at 5000 X G to pellet the thylakoids and resuspending the pellet in a lesser but more appropriate amount of Washing Buffer. NOTE: DO NOT DO THIS DILUTION WITH THE ACETONE! After adjusting the chlorophyll concentration of the thylakoid suspension, remeasure the final chlorophyll concentration in mg chl/mL as described above and record your results. You will use this thylakoid suspension in the subsequent experiments.

The isolated thylakoids in the suspension can now be observed by microscopy by placing a 5 uL aliquot of the 0.5 mg chl/mL suspension on a slide, covering the chlorophyll suspension with a cover slip and observing at 100X with an oil immersion lens. Your instructor will help you photograph your thylakoid membranes at the Nikon microscope in PA 207. The image should be saved as a .jpg or .gif file on a 3.5 inch floppy disk. You can print a picture of your image by pointing a web browser at the the .jpg or .gif file and when you see the image in the browser, using the browsers printing cabability. See an example of thylakoid membranes.

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Isolate physiologically active thylakoid membranes