Western blot and protein expression
Total maize leaf protein was extracted using 0.5 g leaf sample with the
4 mL of PEB buffer (100 mM Tris-HCl pH 6.8, 13.8 mM SDS, 1.43% β-ME
(v:v), 1 mM PMSF, 20 mM NaF, 20% Glycerol (v:v)) after grinding samples
in liquid nitrogen. The mixture was centrifuged at 18000 g for 10 min,
and the supernates were precipitated at -20 °C for 2 h using an equal
volume of pre-cooled acetone. The protein in the precipitate was washed
with 80% pre-cooled acetone at 4 °C for 5 times by centrifugation at
15,000 g for 5 min. Finally, the extracted protein was dissolved in 1 mL
of 1×PBS (pH 7.4 ) containing 5 μL of protease inhibitor cocktail. The
protein was immediately heated at 80 ºC for 5 min and separated on 10%
Tri-glycine SDS-PAGE and blotted 1h to PVDF using semi-dry or tank
transfer. Blots were blocked with 5% non-fat milk in TBST for 1h at
room temperature (RT) with continuous stirring. The blots were then
incubated with primary antibodies (Anti-PHYA, AS07220; Anti-PHYB
AS184170; Anti-PIF4, AS163157; Anti-PIF5, AS122112; Anti-EIN3 AS194273;
Agrisera, Sweden) separately at a dilution of 1: 1,000 overnight at room
temperature with continuous stirring in TBST buffer. After incubation,
the blots were washed for 5 min every 3 times in TBST solution with
continuous stirring. The blots were then incubated with secondary
antibody (Goat anti-Rabbit IgG (H&L), HRP conjugated, AS09602,
Agrisera, Sweden) diluted to 1:10,000 in TBST buffer for 1 h at room
temperature with continuous stirring. The blot was washed in the same
way as described for primary antibody washing. The blots were then
developed for 5 min in a chemiluminescent detection reagent and the
respective bands were observed.