Plant Cell Rep (2009) 28:1747–1758 presence of acid washed sand (0.25 g sand per g cells) for 45 min in a buffer containing 250 mM sucrose, 7 mM citrate buffer pH 5.5 and 5 mM magnesium chloride (MgCl2) (2.5 vol per gram fresh weight of cells). The crude homogenate was ﬁltered through two layers of nylon cloth and the ﬁltrate was centrifuged at 10,0009g for 30 min using a Kubota RA 400 rotor. The supernatant was then centrifuged at 110,0009g for 45 min using a SW 41 Ti swing-out rotor to obtain the microsomal pellet. The microsomes were resuspended in binding buffer containing 7 mM citrate and 5 mM MgCl2 pH 5.5 and dialysed overnight against 0.7 mM citrate buffer and 0.5 mM MgCl2 with four changes of buffer. The dialysed samples were centrifuged at 10,0009g to eliminate any precipitate and used to determine binding of radioactively labelled auxin in the ﬁlter assay. Auxin-binding assay The assay was performed in the dark at 4 C in a ﬁnal volume of 160 ll solution containing 7 mM citrate buffer pH 5.5, 5 mM MgCl2, 83 nM 3H-IAA, 20 ll of micro- somal proteins (10 lg). The samples were incubated for 30 min and the bound radioactivity was displaced by incubating with increasing amounts of non-radioactive IAA (1 nM–100 lM) for 30 min on ice. After the incubation, 44 ll of the reaction mixture were ﬁltered through cellu- lose acetate ﬁlters under vacuum. The ﬁlters were washed with 20 ml washing buffer (0.7 mM citrate buffer pH 5.5 and 0.5 mM MgCl2), dried and the radioactivity retained on the ﬁlters was determined using PPO (poly p-phenylene oxide), and POPOP [1, 4-bis (5-phenyloxazol-2-yl) ben- zene], a toluene based scintillant. Each concentration was tested in triplicate and the mean value was plotted against the radioactivity retained on the ﬁlter. Sub-cellular fractionation for immuno-detection of ABPs and electron microscopy analysis The sub-cellular fractionation of the crude extract was performed under cold conditions. Five grams of frozen moss protonema or corn coleoptiles were homogenised, suspended in grinding buffer [GB: 250 mM sucrose, 20 mM (2-Amino-2-hydroxymethyl-propane-1,3-diol) Tris–HCl pH 8.0, 1 mM ethylenediaminetetraacetic acid (EDTA), 1 mM dithiothreitol (DTT)] with a mixture of protease inhibitors, antipain, leupeptin, chymostatin, pep- statin and tosyl phenylalanyl chloromethyl ketone (TPCK) at a ﬁnal concentration of 50 lM and kept in ice for 15 min. The homogenates were ﬁltered through two layers of nylon cloth and the ﬁltrate was further fractionated by differential centrifugation. Successive centrifugation of the ﬁltrates at 5,0009g for 10 min, 35,0009g for 30 min and 1749 110,0009g for 1 h, resulted in three pellets (P1, P2, P3) and a supernatant (S), respectively. The pellets were washed two times with GB and resuspended in the GB containing 1% sodium cholate and kept for 30 min on ice with occasional vortexing and centrifuged at the respective g forces to eliminate the particulate insoluble material. The resulting supernatant was used in the experiments. To isolate the total solubilised protein, sodium cholate was added dropwise to the ﬁltrate in order to obtain a ﬁnal concentration of 1%. This suspension was further incu- bated for 30 min and centrifuged at 35,0009g for 15 min to extract and use the supernatant. The protein content was estimated using the Bradford dye-binding assay, denatured by boiling in Laemmli sample buffer containing 62 mM Tris pH 6.8, 10% Glycerol, 2% SDS, 0.001% Bromophenol blue and 5% b-mercaptoethanol, separated using a 12% Tris–Glycine gel (Laemmli 1970) and transferred to a nitrocellulose membrane. The P2 and P3 sub-cellular pellet fractions were further processed for transmission electron microscope using a method as described in Shanbhag et al. (1995). The P1 fraction was not processed for EM because at that g forces one would enrich nuclei and mitochondria and other broken contaminants. Immunological detection of ABPs The western blotting and detection was done essentially as described by Harlow and Lane (1988), with few modiﬁ- cations. The Western blots were incubated in 5% fat-free milk in a buffer containing 10 mM Tris–HCl pH 7.5, 50 mM sodium chloride (NaCl), 2 mM calcium chloride (CaCl2) and 0.1% Tween 20 [milk-Tris Buffered Saline- Tween (TBS-T)] for 2 h at room temperature in order to block the non-speciﬁc binding sites. The primary poly- clonal anti-ABP1 or monoclonal anti-KDEL or anti-HDEL antibodies were incubated in milk-TBS-T for 2 h at room temperature or overnight at 4 C on a shaker. Blots were washed four times, with milk-TBS-T and incubated with biotinylated anti-rabbit IgG (for anti-ABP1) or anti-mouse or rat IgG (for anti-KDEL and anti-HDEL) for 2 h at room temperature. Blots were developed using the ABC kit with di-aminobenzidine as a substrate (Vector laboratories, Burlingame, USA). Preparation of extracts for photo-afﬁnity labelling of ABPs The microsomal membranes were prepared from the cal- cium promoted sedimentation of vesicles as described (Shimomura et al. 1986). The microsomal pellet (30 mg dry acetone powder) was suspended in 425 ll of chilled photo- afﬁnity labelling buffer containing 10 mM citrate buffer pH 5.7, 5 mM MgCl2, 1 mM phenylmethanesulphonylﬂuoride 123