CPN60A1 | Chaperonin 60 subunit alpha 1, chloroplastic
AS12 2613 | clonality: polyclonal | host: rabbit | reactivity: A. thaliana, N. tabacum, P. vulgaris, P. sativum, Z. mays
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|Recommended dilution||1 : 1000 (WB)|
|Expected | apparent MW||
57.1 kDa (mature protein)
|Confirmed reactivity||Arabidopsis thaliana, Arabidopsis thaliana cell culture, Nicotiana tabacum, Phaseolus vulgaris, Pisum sativm, Zea mays|
|Predicted reactivity||Aegilops squarrosa, Avicena marina, Brassica napus, Canavalia lineata, Narcissus pseudonarcissus, Oryza sativa, Ricinus communis, Trifolium pRatense, Triticum aestivum|
|Not reactive in||Cyanobacteria, algae|
to be added when available, antibody released in September 2013.
Approximately 50-70 µg of total chloroplast or cell protein was extracted from various species by boiling in 4x Sample buffer for 5 min. These proteins were separated on 15 % Tris-Glycine SDS-PAGE run at constant voltage of 100V for 20min and then run at constant current of 15 mA for 1 hrs. Following separation, the proteins were transferred by electroblotting to PVDF (1h 30 min) using 1X Transfer buffer (14.4 gm glycine, 3 gm Tris-base, 200 ml MeOH in 1l ddH2O) pH 8.3. Blots were blocked with TBS with 1% Tween and 3% NFM (TBST w/NFM)) for 1h at room temperature (RT) with agitation. Blot was incubated in the primary antibody at a dilution of 1: 1000 for 1h at RT with agitation, and then left in 4°C overnight. The antibody solution was decanted and the blot was rinsed briefly three times, and then washed 3 times X 10 min in TBST w/NFM at RT with agitation. The blot was incubated in secondary antibody (Donkey anti-rabbit IgG HRP-conjugated) diluted to 1:15 000 in TBST for 1h at RT with agitation. The blot was washed as above and developed using Thermo SuperSignal West Pico Chemiluminescent Substrate reagent according to the manufacturer’s instructions and imaged on a Bio-Rad ChemiDoc Imager using an exposure time of 80 seconds.
Courtesy of Dr. Barry Bruce lab, University of Tennesee-Knoxville, USA
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