Tic40 | Inner envelope membrane translocon complex protein (chloroplast)
AS10 709 | Clonality: Polyclonal | Host: Rabbit | Reactivity: A.thaliana , N. tabacum, C. roseus, O. sativa, P. patens, S. lycopersicum, V. vinifera cellular [compartment marker] of chloroplast inner envelope membrane
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48 | 45 kDa (Arabidopsis thaliana)
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10-15µg of chlorophyll from isolated total plant material (Arabidopsis thaliana), chloroplasts and thylakoids extracted with a buffer containing (25 mM Tricine-NaOH, pH 7.8, 330 mM sorbitol, 1 mM EDTA, 10 mM KCl, 0.15% [w/v] bovine serum albumin, 4 mM sodium ascorbate, and 7 mM L-Cys) were separated on 12 % SDS-PAGE and blotted 1h to PVDF using semi-dry transfer. Blots were blocked with 10% milk for 1h at room temperature (RT) with agitation. Blot was incubated in the primary antibody at a dilution of 1: 2 000 overnight at 4⁰C with agitation. The antibody solution was decanted and the blot was rinsed briefly twice, then washed once for 15 min and 3 times for 5 min in TBS-T at RT with agitation. Blot was incubated in secondary antibody (anti-rabbit IgG horse radish peroxidase conjugated, from Agrisera AS09 602) diluted to 1:15 000 in TBS-T for 1h at RT with agitation. The blot was washed as above and developed for 60 seconds with a ImageQuant system from GE Healthcare, exposure time was 60 seconds.
Courtesy of Dr. Rikard Fristedt VU University Amsterdam Faculty of Sciences Department of Physics and Astronomy Biophysics of Photosynthesis, The Netherlands
Lanes 1-6: 20μg 21kxg pellet from whole Physcomitrella patens protonemata extract (1), 20μg 21kxg supernatant from whole protonemata extract (2), 20μg broken chloroplasts/thylakoids from 40% Percoll band (3), 20μg intact chloroplasts from 80% Percoll band (4), 10μg pellet material from Percoll gradient (including whole cells and enriched for nuclei) (5), 20μg whole protonemata extract (6)
Sample Preparation Up to 20µg of total protein/chloroplast material from P. patens was prepared by chopping the protonemata into an ice cold isotonic chloroplast buffer (0.3M D-sorbitol, 50mM Hepes-KOH pH8.0, 2mM EDTA, 1mM MgCl2 with additives: 0.1% BSA and PVP-10 and added protease inhibitor tablet). The extract was filtered through a 70μm filter and chloroplasts were pelleted at 250 xg 4°C 5 min then layered onto discontinuous 10, 40, 80% Percoll gradients (which employed the same buffer) and centrifuged at 9k xg 4°C 1h. Broken chloroplasts/thylakoids and chloroplasts, respectively, were recovered from the 40 and 80% bands after centrifugation at 9k xg 4°C 1h by centrifugation at 1k xg in four volumes of buffer (without additives). Samples were frozen at -20°C. Samples were thawed on ice and were diluted in 50mM Tricine pH7.6, 1mM β-mercaptoethanol, 1mM MgCl2 with protease inhibitor tablet for protein concentration determinations and aliquots of up to 20 µg were prepared in 0.8 x Laemmli sample buffer and denatured at 95°C 5 min and were separated on 10-20% Criterion stain free SDS-PAGE and blotted 7 min to PVDF using BioRAD mini Turbo blot semi-dry transfer. Blots were blocked O/N at 4°C with agitation in 10% milk powder in PBS. Blot was rinsed in PBS-T 3 times for 5 min thenincubated in the primary antibody at a dilution of 1: 1 000 for anti-Toc75 or 1 in 2000 for antiTic40 for 2h at RT with agitation in 0.5% milk powder in PBS-T. The antibody solution was decanted and the blot was rinsed briefly twice, then washed once for 15 min and 3 times for 5 min in PBS-T at RT with agitation. Blot was incubated in secondary antibody (anti-rabbit IgG horse radish peroxidase conjugated, from an unknown source but labelled NIF824) diluted to 1:2,500 in for 1h at RT with agitation in 0.5% milk powder in PBS-T. The blot was washed as above and developed for 10min with chemiluminescent detection reagent. Exposure time was 30 min.
Dr. Amanda Dowson, University of Warwick, UK
This product can be sold containing ProClin if requested
Tic40 is a component of the inner envelope membrane import complex (TIC) of plant chloroplasts. Tic40 has been proposed to function as a co-chaperone in the stromal chaperone complex that facilitates protein translocation across the inner membrane. Tic40 can be used as a cellular [compartment marker] for chloroplast inner envelope membrane.
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