As13 2705

For native PAGE, thylakoids were solubilized with 1% digitonin as previously described (Suorsa et al., 2015 (link)), separated with large pore blue native (lpBN)-PAGE as previously described (Järvi et al., 2011 (link)), and either stained with Coomassie brilliant blue or electroblotted to a polyvinylidene difluoride (PVDF) membrane. For one-dimensional SDS-PAGE, thylakoids were solubilized, separated by gel electrophoresis with 15% (w/v) polyacrylamide and 6 M urea and subsequently electroblotted. For 2D gels, the second dimension was run for 3 h at 200 V for an additional ‘3D’ dimension, and the band corresponding to PSII monomer/cytochrome b₆f (Cyt b₆f) was immediately placed on top of a second BN gel with a gradient of 7.5–9.5%.
The antibodies used for immunoblotting were Lhcb1, Lhcb2, P-Lhcb1, P-Lhcb2, CP47, PSB33, STN7 (Agrisera; catalogue numbers AS09 522, AS01 003, AS13 2704, AS13 2705, AS04 038, AS12 1852 and AS10 1611) and TAP38. The TAP38 antibody was a kind gift from Prof. Roberto Barbato. Immunodetection was performed according to standard procedures, with horseradish peroxidase-linked secondary antibody and enhanced chemiluminescence reagents (Amersham, GE Healthcare) used for detection.

The isolated thylakoids were solubilized with a solubilization buffer [138 mM Tris/HCl pH 6.8; 6 M urea, 22.2% glycerol (v/v) and 4.4% sodium dodecyl sulfate (SDS)] containing 10% β-mercaptoethanol, and 0.5 µg of Chl was loaded on SDS-PAGE (12% w/v polyacrylamide and 6 M urea) and separated under a constant current of 10 mA. The proteins were electroblotted to polyvinylidene difluoride (PVDF) membranes and immunoprobed with pLhcb1 and pLhcb2 antibodies (Agrisera AS13 2704 and AS13 2705, 1:10 000 dilution in 1% BSA).

To solubilize the thylakoid protein complexes from the thylakoid samples isolated with or without 5 mM MgCl₂, the samples were diluted in BTH buffer [25 mM Bis/Tris/HCl (pH 7.0), 20% (w/v) glycerol, 0.25 mg/ml Pefabloc and 10 mM NaF]. An equal volume of 2% digitonin (Merck, Calbiochem) in BTH buffer was added to the sample to achieve a final concentration of 0.5 mg ml⁻¹ of Chl and 1% digitonin in the sample and separated with BN-PAGE as described previously (Rantala et al. 2018b ). To investigate the distribution of Lhcb2 proteins in different pools of LHCII trimers, 2D-BN-BN-PAGE was performed as described in (Rantala et al. 2018a ). The 2D-BN gels were electroblotted to PVDF membranes and immunoprobed with pLhcb1 and pLhcb2 antibodies (Agrisera AS13 2704 and AS13 2705, 1:10,000 dilution in 1% BSA) and with Lhcb1, Lhcb2 and Lhcb3 antibodies (Agrisera AS01 004, AS01 003 and AS01 002). (1:2,000–1:5,000 dilution in 1% BSA).