4.6. SDS-PAGE Electrophoresis and Western Blot

The effect of growth at low temperature and high light intensity on PGR5 and PTOX relative abundance was evaluated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblot analysis. For the analysis a Laemmli SDS-PAGE system [74] was used—the polyacrylamide concentrations of the stacking and resolving gels were 4 and 12%, respectively. Urea (4 M) was added to the resolving gel. The samples were incubated with a sample buffer (3:1) in the dark for 1 h at room temperature. Thylakoid membranes that were isolated from wt and lut2 plants before start of treatment (0-day) and from plants grown under double stress for 2 and 6 days and after 7 days of recovery at control conditions were used [75]. Equal amounts of thylakoid membranes corresponding to 3 µg chlorophyll were loaded in every line. The proteins were transferred from SDS-PAGE to polyvinylidene difluoride (PVDF) membrane and proteins were probed with antibodies for PGR5 (AS16 3985—dilution 1:1000) and PTOX (AS16 3692—dilution 1:3000) (Agrisera, Vännäs, Sweden). Development of the blocked membrane was performed using an Alkaline Phosphatase Conjugate Substrate Kit (Bio-Rad, Hercules, California, USA) using a GAR secondary antibody. Densitometric scanning and analysis of each replicate immunoblot was performed with ImageJ 1.41o densitometry software (Wayne Rosband, National Institute of Health, USA, http://rsb.info.nih.gov accessed on 19 July 2022), as described earlier [76]. The presented data were normalized to the relative abundance of PGR5 or PTOX in the control, non-treated plants (0d).