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Fig. 5 | Plant Methods

Fig. 5

From: Qualitative and quantitative evaluation of thylakoid complexes separated by Blue Native PAGE

Fig. 5

Characterisation of untreated maize mesophyll (M) and bundle sheath (BS) thylakoid complexes. a BN PAGE and b CN PAGE separation of complexes from M (a) and BS (a, b) thylakoids. c, d Polypeptide patterns of BS thylakoid preparations of different purity (more or less successful separation from M thylakoids, see the Chl a/b ratios given below). PSI megacomplexes (PSI-mc) are numbered, other complexes are marked as in Fig. 1a. PSII supercomplexes are marked with points. The polypeptides of the NDH complex are numbered (c), and their presence in PSI megacomplexes is marked by arrows. A: pnsB1 (see Table 1) present in PSI megacomplexes, but not present in the free NDH complex. e Two-dimensional BN/BN PAGE of BS thylakoids. PSII supercomplexes are marked with points, and NDH complexes separated in the second dimension BN PAGE by arrows. C2 and C are PSII-d and PSII-m. f Polypeptide pattern (numbered 1–10) of the NDH complexes isolated in the zone marked by arrows in e, and obtained by SDS PAGE. Rubisco L and S subunits are also seen in the right side of the pattern. g Amounts of the Ndh polypeptides non-overlapping with PSI ones in PSI megacomplexes normalised to PSI core 4–6 (see in c). Differences in each polypeptide between PSI-mc1 and PSI-mc2 were compared using multiple t-tests [P < 0.05; n = 3 × 2 (biological × technical)]. Significant differences are marked with stars (*). The Chl a/b ratio of the used M and BS (a, b), BS1 (c, e), and BS2 (d) thylakoids were 3.62, 6.27, 6.97, and 5.95, respectively. Thylakoids (500 µg Chl mL−1) were solubilised using 1% (w/V) β-DM plus 1% (w/V) digitonin in the first-dimensional BN/CN PAGE, and with 1% (w/V) β-DM in the second-dimensional BN PAGE. Complexes were separated in 4.3–12% (ac, g—first dimension, e—second dimension) and 4.3–8% (d, e—first dimension) BN/CN gel gradients followed by SDS PAGE to obtain the polypeptide patterns of bands (c, d, f, g). Standard proteins: PageRuler™ Plus Prestained Protein Ladder (ThermoFisher Scientific 26619, Lot #00803392)

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