Thioredoxin-like protein HCF164, chloroplastic
AS16 4097 | Clonality: Polyclonal | Host: Rabbit | Reactivity: Arabidopsis thaliana
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|Recommended dilution||1 : 1000 (WB)|
|Expected | apparent MW||
28.6 | 26 kDa
|Confirmed reactivity||Arabidopsis thaliana|
|Predicted reactivity||Aegilops tauschii, Anthurium amnicola, Cajanus cajan, Cephalotus follicularis, Cicer arietinum, Corchorus olitorius, Cucumis melo, Dichanthelium oligosanthes, Glycine soja, Gossypium hirsutum, Medicago truncatula, Morus notabilis, Musa acuminata, Nelumbo nucifera, Nicotiana sylvestris, Nicotiana tabacum, Oryza sativa subsp. japonica, Saccharum hybrid cultivar R570, Solanum chacoense , Theobroma cacao, Vigna radiata var. radiata, Zea mays|
|Not reactive in||No confirmed exceptions from predicted reactivity are currently known.|
To be added when available, antibody available in September 2017.
Isolated thylakoids from Arabidopsis thaliana were extracted with grinding buffer containing 300mM Sucrose, 50 mM Hepes-NaOH pH 7,4, 5 mM MgCl2, 1 mM Na-EDTA containing freshly made 10 mM NaF, then the chloroplast pellet was dissolved with shock buffer containing, 5 mM Sucrose, 10 mM Hepes-NaOH pH 7,4, 5 mM MgCl2 containing freshly made NaF (10 mM), whereafter pellet containing thylakoid fraction was carefully again dissolved in to the storage buffer containing 100mM Sucrose, 10 mM Hepes-NaOH pH 7,4, 10 mM MgCl2 containing freshly made 10 mM NaF. Proteins were denatured with Laemmli buffer at 65C for 5 min. Protein amounts loaded according to PORRA corresponding to 1,2, or 4µg of Chlorophyll from thylakoids were separated on 12 % Acryl Amide containing 6M Urea with SDS-PAGE and blotted 1h to PVDF membrane using semi-dry transfer (Hoefer TE77X). Directly after protein transfer, membrane was washed 1 min in 100% Methanol followed by rinsing in ddH2O and 1xTBS for 2 min. Membranes were then blocked with 4% Milk in 1XTBS for 1h at room temperature (RT) with agitation. Blot was washed then 3x5min 1xTTBS, and incubated in the HCF164 primary antibody at a dilution of 1: 3 000 for 24h at +4C with slow agitation in 1% Milk in 1XTTBS. The antibody solution was decanted and the blot was rinsed 5 minutes 3 times each with TTBS at RT with agitation. Blot was incubated in secondary antibody 800CW diluted to 1:25 000 in TTBS in the presence of 0.01% SDS (IRDye® 800CW Goat anti-Rabbit IgG, LiCor) for 1h at RT with agitation in the dark. The blot was washed as above, with additional washes of 2X4 min in 1XTBS. Membrane was dried 20 minutes in foil covered plastic box in the hood in darkness keeping box lid slightly open until membrane is completely dry, then followed by signal detection at near-infrared fluorescence scanning at 800 nm with LiCor Odyssey CLX. Data was handled with Licor Odyssey software and membrane was stored dried in RT dark.
Courtesy of Dr. Eevi Rintamäki, University of Turku, Finland
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