CGL78 | YCF54
AS10 936 | clonality: polyclonal | host: rabbit | reactivity: A. thaliana, Ch. reinhardtii, Synechocystis sp. PCC6803
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1 : 1000 with standard ECL (WB)
|Expected | apparent MW||
Arabidopsis thaliana , Chlamydomonas reinhardtii, Synechocystis sp. PCC 6803
|Not reactive in||
Please, omitt SDS from transfer buffer and reduce transfer time to 45 min. Nitrocellulose membrane is recommended and SDS is omitted to allow this LMW protein to bind tighter to the membrane.
Hsieh et al. (2013). The Proteome of Copper, Iron, Zinc, and Manganese Micronutrient Deficiency in Chlamydomonas reinhardtii. Mol Cell Proteomics. 2013 Jan;12(1):65-86. doi: 10.1074/mcp.M112.021840. Epub 2012 Oct 13.
15 µg of total protein from Arabidopsis thaliana (ecotype Col-0), Chlamydomonas reinhardtii (strain 4A+) and Synechocystis sp. (strain PCC6803 / Kazusa), extracted with 56 mM Na2CO3, 56 mM DTT, 1 % (w/v) SDS, 12 % (w/v) Sucrose, 2 mM EDTA) were separated on 15% SDS-PAGE and blotted 1h to nitrocellulose membrane. Blot was blocked with 2% milk powder in TBS-T for 1h at room temperature (RT) with agitation. Blot was incubated in the primary antibody at a dilution of 1:1000 for 1h 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 (anti-rabbit IgG horse radish peroxidase conjugated, from Sigma) diluted to 1:20,000 in 1% milk powder in TBS for 1h at RT with agitation. The blot was washed as above and developed for 5 min with ECLTM Western Blot Detection Reagent (GE Healthcare, Freiburg, GER) according to the manufacturers instructions. Exposure time was 3 minutes.
Courtesy of Dr. Annabel Salinas Hartwig, Humboldt University, Germany
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