类囊体膜的提取

浓度名称分子量
捣碎液 : PH 7.8 2500ml 5000ml 1000ml
20 mM Tris-HCl [121.14] (6.057g) （12.114） 2.4228
0.4 M Sucrose [342.3] (342.3g) (684.6) 136.92
15 mM NaCl [58.44] (2.192g) (4.383) 0.8766
2 mM EDTA二钠 [372.2] (1.861g) (3.722) 0.7444
胀破液：PH 7.8 1000ml 2000ml
20 mM Tris-HCl [121.14] (2.423g) (4.846)
[203.3] (1.017g) (2.033)
5 mM MgCl
2
15 mM NaCl [58.44] (0.8766g) (1.753)
保存液：PH 7.0 1000ml 2000ml
20 mM Tris-HCl [121.14] (2.423g) (4.846)
35 mM NaCl [58.44] (2.045) (4.09)
0.4 M Sucrose [342.3] (136.92g) (273.84)
Activities of electron transport: Thylakoid membranes were isolated from the leaves as described by Berthold et al. (1981). Whole chain electron transport (H2O→methyl viologen, MV) and partial reactions of photosynthetic electron transport mediated by PS2 (H2O→2,6- dichloro-p-benzoquinone, DCBQ; H2O→sil icomolybdate, SiMo) and PS1 (DCPIPH2→MV) were measured as described by Nedunchezhian et al. (1997). Thylakoids were suspended at 10 mg(Chl) m-3 in the assay medium containing 20 mM Tris-HCl, pH 7.5, 10 mM NaCl, 5 mM MgCl2, 5 mM NH4Cl, and 100 mM sucrose supplemented with 500 μM DCBQ and 200 μM SiMo.
类囊体的提取步骤：
Highly active intact thylakoid membranes were prepared as in [6]. Subchloroplast membranes capable of oxygen evolution were prepared by suspension of the intact thylakoids (2 mg chl/ml) in MgC12 (5 mM), NaCl(15 mM) and Hepes buffer (20 mM, pH 7.5) and incubation with Triton X-100 (25 mg/mg chl) at 4°C for 30 min. The fraction of chl-containing material sedimenting at 40 000 X g (30 min) was resuspended in incubating buffer (2 mg chl/ml) with Triton X-100 (5 mg/mg chl), recentrifuged immediately (40 000 X g, 30 min) and stored in sucrose (0.4 M), MgClz (5 mM), NaCl(15 mM) and Hepes (20 mM, pH 7.5) at -35°C for subsequent analyses. In this procedure, which is superficially similar to that in [3], we have produced a set of conditions with regard to salt concentrations whereby O2 evolution activity is resistant to denaturation by exposure to Triton X-100. Procedures for assay of oxygen evolution, for Tris-inhibition, and EPR detection of signals II, and IIf have been reported in [7,8]. Cytochrome content was assayed optically by using an Aminco DW-2 spectrophotometer.
Assay of pigments content
Leaf tissues were homogenized in chilled N, N-dimethylformamide using a mortar and pestle in dark at 4 °C, and the homogenates were centrifuged at 8,800 × g for 10 minutes. The supernatants were collected and the absorption spectra at 663 and 646 nm were recorded for the estimation of chlorophyll a (Chl a) and chlorophyll b (Chl b) following the procedure described by Inskeep and Bloom (1985). For the
estimation of total carotenoids (Car), leaf tissues (300 mg) were homogenized in chilled 80% (v/v) acetone, and then centrifuged at 8,800 × g for 10 minutes in dark at 4 °C. The absorbance of the acetone extracts was measured at 663, 646 and 470 nm. Total carotenoids content was calculated as described by Wellburn (1983).。