GPA1 | Guanine nucleotide-binding protein subunit alpha 1 (affinity purified)
AS12 2370 | Clonality: Polyclonal | Host: Rabbit | Reactivity: Arabidopsis thaliana
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25 µg of total protein from the indicated Arabidopsis thaliana wilde type and gpa1-3 mutant were extracted with extraction buffer ( 250 mM sucrose, 100 mM HEPES-KOH pH 7.5, 5% glycerol, 1mM Na2MoO4 x 2H2O, 25mM NaF, 10 mM EDTA, 1mM DTT, 0.5% Triton X-100, protease inhibitor cocktail) and denatured with SDS loading dye (50 mM Tris-HCl pH6.8, 100mM DTT, 2%SDS, 10% glycerol, 0.025% bromophenol blue) at 70°C for 2-5 min were separated on 10% SDS-PAGE and blotted 1h to PVDF using tank transfer. Blots were blocked with Tris-buffered saline containing 0.05% Tween-20 (TBS-T) and 5% skimmed milk powder for 1h at room temperature (RT) with agitation. Blot was incubated in the primary antibody (anti-GPA1, AS12 2370) at a dilution of 1: 5 000 overnight at 4°C with agitation. The antibody solution was decanted and the blot was rinsed briefly twice, then washed 5 times for 15 min in TBS-T with milk powder at RT with agitation. Blot was incubated in secondary antibody (Goat-Anti-Rabbit HRP conjugate, AS09 602) diluted 1:5000 for 2h at RT with agitation. The blot was washed as above with TBS-T without milk powder and then developed with Thermo Scientific SuperSignal West Substrate (pico and femto mixed 1:1), before exposure to a CEA RP NEW film for 10min.
Courtesy of Dr. Elena Petusching, Georg-August-University Goettingen, Germany
Immunodetection of GPA1 (alpha subunit of heterotrimeric G protein). Microsomes were affinity purified to isolate palmitoylated proteins using the acyl biotin exchange method1. Proteins were separated by SDS-PAGE and immunodetected with anti-GPA1. GPA1 is expected to be modified by S-acylation and was enriched in the affinity-purified fraction.
1. Wan, J., Roth, A. F., Bailey, A. O. & Davis, N. G. Palmitoylated proteins: purification and identification. Nat. Protoc. (2007). doi:10.1038/nprot.2007.225
Palmitoylated (S-acylated) proteins were affinity purified from Arabidopsis thaliana using the acyl biotin exchange method1 and separated using SDS-PAGE. Proteins were electro-transferred to PVDF membrane (Millipore, Cat. No. IVPH00010). The membrane was blocked with 5% non-fat dry milk (NFDM) in Tris-buffered saline (TBS) for two hours at room temperature. All incubations were performed with gentle agitation. The membrane was treated with primary antibody (anti-GPA1 [Agrisera, Cat. No. AS12 2370]; 1:7500 in TBS containing 5% NFDM and 0.05% Tween-20) for 2 hours. Blots were quickly rinsed twice with TBS containing 0.05% Tween (TBS-T), then washed five times for 15 minutes each with TBS-T on a shaker. The membrane was treated for 45 minutes with HRP-tagged secondary antibody (Agrisera, Cat. No. AS09 602) diluted 1:10 000 in TBS-T containing 5% non-fat dried milk. Blots were washed 2-3 times for 15 minutes with TBS-T, followed by two 15-minute washes with TBS. Immunolabeled proteins were detected with chemiluminescent detection reagent and imaged using X-ray film.
Courtesy of John McLarney under the guidance of Dr. Estelle Hrabak, University of New Hampshire, USA
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