Experimental proteomic analysis of Arabidopsis thaliana chloroplasts
This chapter describes a simple and practical method for large-scale isolation and purification of chloroplasts. The advantages of this method are high yield, low contamination and less degradation of proteins. The isolated chloroplast samples are suitable for proteomic analysis. The source for this experiment is the "Guide to Plant Proteomics Experiments" [French] H. Tillmant, M. Zivi, C. Damerwell, V. Mitschine, eds.
Operation method
Proteomic analysis of Arabidopsis thaliana chloroplasts
Materials and Instruments
HEPES-KOH Sorbitol Ascorbic Acid Vitamin C Cysteine PF-Percoll Move It is recommended to incubate the material under short sunlight conditions to induce nutrient growth and to collect the material at an early stage of illumination to increase the yield of obtaining intact chloroplasts. Therefore, the reagents should be prepared before collecting the material, and together with some other equipments, such as centrifuge head and centrifuge tubes, etc., should be cooled down in refrigerator or on ice to 0~4°C. In order to avoid excessive exposure of chloroplasts to the light or fragmentation, all the operations should be completed as quickly as possible under green light. The whole separation process can be completed in 30~40 min. For more product details, please visit Aladdin Scientific website.
Concentration and centrifugation equipment
( 1 ) Cultivate Arabidopsis material under 10 h light (23°C) / 14 h dark (17°C) photoperiodic conditions for 5-6 weeks, and collect the leaves before flowering.
( 2 ) Prepare all the solutions and cool them down to 0-4°C together with all the equipments (including centrifuge and rotary head).
( 3 ) Cut plant leaves (see Note 2) and wash with distilled water to ensure that no soil adheres.
( 4 ) Mix the material and pre-cooled buffer at a ratio of 100 ml of homogenization buffer (Medium A) per 10 g of material and grind in a blender with a sharp blade 3 times for 10 s each time.
( 5 ) The homogenate is filtered through a double layer of nylon cloth (22 μm, see Note 3).
( 6 ) The filtrate is centrifuged at a centrifugal force of 1300 g for 3 min, and the precipitate is the crude chloroplast extract.
( 7 ) Resuspend the crude chloroplast extract in the smallest possible volume of Medium B (gently stir the liquid to suspend the precipitate). The suspended extract is placed on the surface of a Percoll discontinuous gradient (40%-85%, see Note 4) and centrifuged at 3750 g for 10 min with a swing-out (non-fixed corner) rotor.
( 8 ) Intact chloroplasts are collected from the interface of 40% and 85% Percoll with a pointed pipette, and Medium B is added to dilute the Percoll until the chlorophyll concentration is 1~3 mg/ml (see Note 5). Then centrifuge at 1200 g for 3 min and the resulting precipitate is the intact chloroplasts.
( 9 ) To continue the isolation of cysts and matrix proteins, the chloroplast precipitate is gently suspended in lysis solution to a chlorophyll concentration of 1-3 mg/ml. The suspension is placed on ice for 5-10 min to allow the chloroplasts to absorb water and swell, and ultimately rupture, releasing the matrix proteins. The sample can also be gently homogenized with a micromill to better break up the chloroplasts, and care should be taken not to grind too hard to avoid destroying the cysts.
( 10 ) Centrifuge the sample at 10000 g for 20 min, the supernatant is the matrix protein extract and the precipitate is the cysts. For better removal of contamination, the supernatant should be centrifuged at 300000 g for another 25 min (see Note 6).
( 11 ) Concentrate the matrix proteins using a centrifugal concentrator containing a 3kDa filter. The lysate has a relatively low concentration of various salt ions and is compatible with various sample buffers (e.g., Isoelectric Focusing Buffer, SDS Buffer, Blue-Green or Colorless Non-Denaturing Gel Buffer, and buffers used for proteolytic digestion).
( 12 ) The ascomycete fraction is resuspended in the same lysate and subjected to pigment and protein quantification. Subsequent proteomic analyses require the removal of pigments (chlorophyll and carotenoids) as well as lipid-soluble molecules (e.g., plastoquinone) contained therein with organic solvents (e.g., 70% acetone) or ionic (SDS) and nonionic (OGG, DM ) denaturing agents. Some recent analyses of the chloroplast membrane proteome can be found in the literature [7, 8, 10, 11 , 18, 19].