PsaH | PSI-H subunit of photosystem I, Chlamydomonas

Synechococcus sp. PCC 7942

Application example

2 µg of total leaf protein of Arabidopsis thaliana (1) and Hordeum vulgare (2) and total cellular protein of Chlamydomonas reinhardtii (3) and Synechococcus PCC 7942 (4) isolated with Agrisera Protein Extraction Buffer (AS08 300), were separated on 4-12% Nupage Bis-Tris gels in in MES running buffer (Invitrogen) at 200V for 35 minutes. Proteins were transferred for 80 minutes at 30V to a PVDF membrane pre-wetted in methanol and equilibrated in 1X transfer buffer. Blots were blocked immediately following transfer in 2% blocking reagent in 20 mM Tris, 137 mM sodium chloride pH 7.6 with 0.1% (v/v) Tween-20 (TBS-T) and probed with anti-PsaH (AS06 143, 1:10000) and secondary HRP-conjugated goat anti-rabbit antibody (1:50 000) for 1 hr in TBS-T containing 2% blocking reagent. Antibody incubations were followed by washings in TBS-T (15, +5, +5, +5 min). All steps were performed at RT with agitation. Signals was detected after 3 s using chemiluminescent detection reagent (GE Healthcare) according to the manufacturers instructions and a CCD imager (FluorSMax, Bio-Rad).

PsaH (PSI-H) is a conserved subunit of type I photosynthetic reaction centers (Photosystem I, PSI). PSI is an integral membrane multi-protein complex that catalyzes the electron transfer from plastocyanin (or cytochrome c6) to ferredoxin (or flavodoxin). Psa-H has been suggested to be involved in regulation of state1-state2 transitions. In plants and green algae Psa-H is nuclear encoded and imported post-translationally into the chloroplast where it inserts into the thylakoid membrane.