DnaK | chloroplast stromal chaperone
AS07 270 | Clonality: Polyclonal | Host: Rabbit | Reactivity: Chlamydomonas reinhardtii, Synechocystis 6803 motile, Synechocystis 6803 GT (glucose tolerant strain), Synechococcus elongates 7942
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recombiant HSP70B of Chlamydomonas reinhardtii (XP_001696432), UniProt: A8HYV3
15 µg of Arabidopsis thaliana leaf extract (1), 10 µg of total protein from: Synechocystis 6803 motile (2), Synechocystis 6803 GT (glucose tolerant strain) (3), Synechococcus elongates 7942 (4), Marker - P ierce™ Prestained Protein MW Marker (kat #26612): Total protein was extracted with following buffer: 10 mM Tris HC l, pH 8.0, 0.5% LDS, 4% glycerol, 0.1 mM EDTA were mixed with sample buffer and denatured for 5 min at 95°C. Samples were separated on 10% S DS -PAGE a nd b lo tted 1 h to nitrocellulose membrane (Amersha m Protran) using tank wet transfer (Bio -Rad) in standard transfer buffer in presence of 20% methanol. Transfer of proteins to the membrane was checked using 0,5% Ponceau S staining before the blocking step. Blots were blocked in buffer (2 % lo w -fat milk in 1xPBS, 0,1% Tween) for 1 h at room temperature (RT) with agitation. Blots were incubated in the primary antibody at a dilution of 1 : 5000 for 1 h at RT with agitation. The antibody solutionwas decanted and the blot was rinsed briefly twice, then washed once f or 15 min and 3 times for 5 min in PBS -T at RT with agitation. Blot was incubated in secondary antibody (goat anti-rabbit IgG, AS09 602, Agrisera ) dilut ed to 1 :30 000 in for 1 h at RT with agitation. The blot was was washed as above and developed for 5 min with Clarity Western ECL Substrate and ChemiDoc detection system (Bio-Rad).
Courtesy Dr. Elena Pojidaeva, Laboratory of Plant Gene Expression, Timiryazev Institute of Plant Physiology RAS, 127276 Moscow Russia
Procaryotic Hsp70 protein family includes DnaK, HscA (Hsc66), HscC (Hsc62). Those proteins are involved in protein folding, cell protection from environmental stress and other functions which are vital for a living cell.
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