Hrp coupled anti rabbit igg

Immunoblot analysis of lysates and chemiluminescence detection was done as described [94 (link)]. Primary antibodies were the antipeptide antibody against AtGRP7 (rabbit; dilution 1:2500), which discriminates AtGRP7 from AtGRP8 and lacks a signal in grp7-1 [21 (link)], a polyclonal serum against LHCP (rabbit; 1:25,000) [95 (link)], and a monoclonal antibody against GFP (Roche catalog number 11 814 460 001; mouse; dilution 1:1000). Secondary antibodies were HRP-coupled anti-rabbit IgG (Sigma-Aldrich catalog number A 0545; dilution 1:5000) or HRP-coupled anti-mouse IgG (Sigma-Aldrich catalog number A0168; dilution 1:2500).

Protein samples were diluted with SDS sample buffer (100 µM Tris/HCl, pH 6.8 with 4% SDS, 0.2% bromphenol blue and 20% glycerol). Prior to separation, samples were boiled for 20 min and loaded onto a SDS-Gel containing 7.5 or 10% acrylamide. PAGE Ruler unstained or prestained protein marker (Fermentas, St. Leon-Roth, Germany) was used as a molecular weight standard. After separation, the gel was stained with Coomassie brilliant blue G250 (Serva, Heidelberg, Germany). For Western blot, samples were separated via SDS-PAGE as described above using PAGE Ruler prestained protein marker (Fermentas, St. Leon-Roth, Germany) and were subsequently transferred onto a PVDF membrane by tank-blotting. After blocking for 1 h in TBST [TBS (pH 7.4) containing 0.1% Tween 20] supplemented with 5% dried milk, the blot was incubated for 4 h with an anti CK2β-serum (polyclonal rabbit serum, raised against amino acids 206–215 of CK2β-subunit, Serum# 32, [27 (link)]), diluted 1:5,000 in TBST, followed by three washing steps with TBST. The membrane was then incubated for 1 h with the secondary antibody [HRP-coupled anti rabbit IgG (#AO545, Sigma-Aldrich, Deishofen, Germany), dilution of 1:10,000 in TBST] followed by two washing steps with TBST and one with TBS. The immune complex was visualized via chemiluminescence (ImmunoCruz kit, Santa Cruz biotechnology, Heidelberg, Germany).

Immunoblot analysis of total lysate, cytoplasmic and nucleoplasmic fractions was essentially performed as described [29 (link)]. Briefly, primary antibodies were polyclonal antibodies against HISTONE 3 (Agrisera, Vännäs, Sweden, AS10710; rabbit; dilution 1:5000), PHOSPHOENOLPYRUVATE CARBOXYLASE (Agrisera AS09458; rabbit; dilution 1:20,000) and SERRATE (Agrisera AS09532A; rabbit; dilution 1:1000). Secondary antibody was HRP-coupled anti-rabbit IgG (Sigma-Aldrich, Burlington, MA, USA, A0545; dilution 1:2500). Chemiluminescence detection of the immunoblots was done with Fusion-FX6 (Vilber) system.