GST-tag (rabbit antibody, polyclonal)
AS17 4147 | Clonality: Polyclonal | Host: Rabbit | Reactivity: GST-tagged proteins
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1: 1: 1000 (ELISA), 1: 2000 (WB)
Samples with 8, 6, 2, 4, 0.5 µg of GST-tagged protein were denatured with Laemmli sample buffer at 70°C for 5 min and separated on 12,5% SDS-PAGE (Amresco). Proteins were blotted 1h to HybondTM-PVDF membrane (GE Healthcare) using semi-transfer (Bio-Rad) in standard transfer buffer in presence of 10% methanol. Transfer of proteins to membrane was checked using 1% Ponceau S staining before the blocking step. Blots were blocked in buffer (5% low-fat milk in 1xTBS, 0.1% Tween) with for 1 h at room temperature (RT) with agitation. Blots were incubated in the primary antibody at a dilution of 1:5 000 1 h at RT 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 (goat anti-rabbit IgG, HRP-conjugated, AS09 602, Agrisera) diluted to 1:25000 (Agrisera) in for 1h at RT with agitation. The blot was washed as above and developed for 5 min with Clarity Western ECL Substrate and ChemiDoc detection system (Bio-Rad).
Courtesy of Dr. Dr. Elena Pojidaeva, Laboratory of Plant Gene Expression Timiryazev Institute of Plant Physiology RAS, Russia
1 - 100 ug of Escherichia coli extract expressing recombinant MBP-tagged PMI1 N-domain after induction with IPTG
2 - 50 ug of Escherichia coli extract expressing recombinant GST-tagged SnRK2.10 before induction with IPTG
3 - 50 ug of Escherichia coli extract expressing recombinant GST-tagged SnRK2.10 after induction with IPTG
4 – 1 ug of purified GST-tagged SnRK2.4
Total protein extract (samples 1,2 and 3) was prepared from E. coli by centrifuging 1ml of bacteria in LB before/after 3h of induction with IPTG for 5 min in 5000 rpm. Supernatants were decanted and pellets were suspended in water. Purified recombinant GST-tagged SnRK2.4 (sample 4) was suspended in Tris-HCl buffer pH 7.5. Samples were then denatured with 3x Laemmli sample buffer for 5 min in 95°C. Next samples were separated on 10 % SDS-PAGE stain-free gels (Bio-Rad) and blotted overnight to PVDF membrane using wet transfer. Blot was blocked with 3% milk and 0,1% Tween 20 in TBS for 1h/RT with agitation. Blot was incubated in the primary antibody at a dilution of 1: 5 000 for 1,5h/RT with agitation in 3% milk and 0,1% Tween 20 in TBS solution. The antibody solution was decanted and the blot was rinsed briefly, then washed 5 times for 5 min in TBS-T at RT with agitation. Blot was incubated in Agrisera matching secondary antibody AS09 602 (goat anti-rabbit IgG horse radish peroxidase conjugated) diluted to 1:30 000 in for 1h/RT with agitation. The blot was washed as above and developed for 1 min with chemiluminescent detection reagent. Exposure time was 2 min.
Courtesy of Dr. Katarzyna Szymańska, Institute of Biochemistry and Biophysics PAS, Warsaw, Poland
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