LEA4-5 | Late embryogenesis abundant protein 4-5 (affinity purified)
AS13 2758A | Clonality: Polyclonal | Host: Rabbit | Reactivity: Arabidopsis thaliana
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1 : 1000 (WB)
|Expected | apparent MW||
|Predicted reactivity||Arachis hypogaea, Brassica sp., Glycine max, Medicago truncatula, Phaseolus vulgaris, Thellungiella halophila|
|Not reactive in||
no confirmed exceptions from predicted reactivity are currently known
to be added when available, antibody released in August 2016.
Protein extracts were obtained from Arabidopsis thaliana seeds (extract), beside recombinant AtLEA4-5 loaded in indicated amounts. Following extraction buffer was used: 0.7 M sucrose, 0.5 M Tris-base, 30 mM HCl, 50 mM EDTA, 0.1 M KCl, 2% β-mercaptoethanol, 12 mg/ml poly-vinyl-poly-pyrrolidone (PVPP). This buffer was complemented with 2mL of equilbrated phenol before extraction. Samples were centrifuged and the protein phase was recovered; the extracted proteins were precipitated with 0.1 m ammonium acetate dissolved in methanol, centrifuged and the pellet washed with cold (-20 ℃) 80% acetone. The protein pellet was dissolved in SDS-solubilization buffer (1% CHES, 2% SDS, 2% ß-mercaptoethanol, 10% glicerol). Thirty 𝜇g of seed protein extract was denatured with Laemmli buffer at 95 °C for 5 min and proteins were separated on 15% SDS-PAGE and blotted 1.5 h to nitrocellulose membrane in a liquid transfer system. Blots were blocked with 2% skim milk ON at 4°C with agitation. After rinsing with TBS, blots were incubated in the primary antibody at a dilution of 1:1 000 (anti-AtLEA4-5) for 3h at 4°C with agitation. The antibody solution was decanted and the blot was rinsed briefly, then washed once for 15 min. and 3 times for 5 min in TBS-T at 4°C with agitation. Then, blots were incubated in secondary antibody (anti-rabbit IgG horse-radish peroxidase conjugate) diluted to 1:25 000 in for 1h at RT with agitation. The blot was washed as above and developed for 2 min with SuperSignal West Pico Chemiluminescent Substrate, Thermo Fisher Scientific. Exposure time was for 30 seconds.
Courtesy of Dr. Alejandra Covarrubias, UNAM, Mexico Courtesy of Dr. Alejandra A. Covarrubias, UNAM, Mexico
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