PsbA | D1 protein of PSII, C-terminal (chicken)

KLH-conjugated synthetic peptide derived from available plant, algal and cyanobacterial PsbA sequences, including Arabidopsis thaliana UniProt: A4QJR4, TAIR: AtCg00020 , Oryza sativa P0C434, Populus alba Q14FH6, Physcomitrella patens Q6YXN7, Chlamydomonas reinhardtii P07753, Synechocystis sp. P14660 and many others

38 | 28-30 kDa

Algae (brown and red), Conifers, Cryptomonads, Legumes, Stramenopiles, Euglenoids, Prochlorophytes, Xantophytes

Application example

2 µg of total protein from (1) Arabidopsis thaliana leaf, (2) Horderum vulgare leaf, (3) Chlamydomonas reinhardtii total cell, (4) Synechococcus sp. 7942 total cell, (5) Anabaena sp. total cell extract. All extracts were extracted with PEB (AS08 300) and separated on  4-12% NuPage (Invitrogen) LDS-PAGE and blotted 1h to PVDF. Blots were blocked immediately following transfer for 1h at room temperature with agitation. Blots were incubated in the primary antibody at a dilution of 1: 50 000 for 1h at room temperature 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 room temperature with agitation. Blots were incubated in secondary antibody (anti-hen IgY horse radish peroxidase conjugated, recommended secondary antibody AS09 603) diluted to 1:50 000 for 1h/RT with agitation. The blots were washed as above and developed for 5 min with chemiluminescent  detection reagent, according the manufacturers instructions. Images of the blots were obtained using a CCD imager (FluorSMax, Bio-Rad) and Quantity One software (Bio-Rad).

A number of degradation products may be observed when using anti-PsbA antibodies, including products having apparent molecular weights of 24kDa and 16kDa. D1 degradation is a complex set of events and the products observed can be influenced by both the extraction procedure and the physiology of the cells prior to harvest. Third, cross-linking may occur between D1 and cytochrome b559, shifting the protein higher in the gel. In cyanobacteria (PCC7942), three different bands were competed out by preincubating the antibody with the PsbA free peptide, indicating that all bands are indeed PsbA and its precursors or breakdown products. Competition assays were also performed with spinach and Chlamydomonas, confirming the identity of PsbA bands. Anti-PsbA antibodies will not detect D2 protein, as the peptide used to generate PsbA antibodies has no homology to the D2 sequence.

Example of a simulataneous western blot detection with RbcL, PsbA and PsaC antibodies.

The antibody is appropriate for detecting both, 24 kDa or the 10 kDa C-terminal fragments, whichever is generated under given treatment conditions.In our analysis we have seen both, ca. 24 kDa and ca. 10 kDa fragments from different samples, depending on treatments and isolation procedures.

This antibody will also detect the phosphorylated form of D1as an alternate band to the main band on a high resolution gel.

The PsbA (D1) protein of Photosystem II is rapidly cycled under illumination in all oxygenic photobionts. Disruption of PsbA cycling or losses of PsbA pools are central to photoinhibition of photosynthesis in cyanobacteria, algae and plants under a wide range of conditions including excess light, low temperature and UV exposure. Tracking PsbA pools using the Global PsbA antibody can show the functional content of Photosystem II in a wide range of samples.