Anti-PsaK | PSI-K subunit of photosystem I

Protein samples corresponding to 1.0 µg chlorophyll of chloroplasts extracted from Arabidopsis thaliana.  Chloroplast samples were isolated with 20 mM Tricine–NaOH, pH 8.4, containing 400 mM sorbitol, 5 mM MgCl2, 5 mM MnCl2, 2 mM EDTA, 10 mM NaHCO3, 0.5% (w/v) bovine serum albumin (BSA) and 5 mM ascorbate: and denatured with exact buffer (62.5 mM Tris-HCl, pH 6.8, containing 2% (w/v) SDS, 10% (w/v) glycerol, 0.005% (w/v) bromophenol blue, and 50 mM dithiothreitol) at 95 °C/5 min.  Samples were separated at RT on 10%-20% gradient gel and blotted for 0.5 h to PVDF (pore size of  0.2  µm), using: semi-dry transfer at RT. Blot was blocked with 5 % milk  for: 1h/RT with agitation. Blot was incubated in the primary antibody at a dilution of 1:  1,000 for 1h/RT with agitation in TBS-T. The antibody solution was decanted, and the blot was rinsed briefly twice, then washed 3 times for 5 min in TBS-T at RT with agitation. Blot was incubated in  matching secondary antibody (anti-rabbit IgG horse radish peroxidase conjugated, AS09 602, Agrisera) diluted to 1: 25,000 in  for 1h/RT with agitation. The blot was washed as above and developed with a following chemiluminescent detection reagent: AS16 ECL-N-10 AgriseraBright (mid picogram). Exposure time was 3 minutes.

Courtesy of Dr. Yuki Okegawa, Okayama University, Japan