Plant/Algal cell antibodies / Chlamydomonas reinhardtii

product information

The psbA gene has been cloned from many species of plants, green algae, and cyanobacteria. The psbA gene is located in the chloroplast genome and encodes for the D1 protein, a core component of Photosystem II. PsbA/D1 is rapidly cycled under illumination in all oxygenic photobionts. Tracking PsbA pools using the Global PsbA antibody can show the functional content of Photosystem II in a wide range of samples.

KLH-conjugated synthetic peptide derived from available plant,algal and cyanobacterial PsbA sequences, including Arabidopsis thaliana AtCg00020, Oryza sativa P0C434, Physcomitrella patens Q6YXN7, Chlamydomonas reinhardtii P07753, Synechococcus sp. P14660

store lyophilized/reconstituted at -20°C; once reconstituted make aliquots to avoid repeated freeze-thaw cycles. Please, remember to spin tubes briefly prior to opening them to avoid any losses that might occur from lyophilized material adhering to the cap or sides of the tubes.

western blot (WB)

AS05 084 | PsbA | D1 protein of PSII, C-terminal

AS01 016 | anti-PsbA C-terminal hen antibody

AS06 124A | anti-PsbA, N-terminal rabbit antibody

AS01 016S | PsbA protein standard for quantitation and positive control

recommended secondary antibody

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.

application information

1: 10 000 with standard ECL (WB)

38 | 28-30 kDa

Arabidopsis thaliana, Hordeum vulgare, Nicotiana benthamiana,Panicum miliaceum, Panicum maximum, Zea mays, Physcomitrella patens, Chlamydomonas reinhardii, Synechococcus sp. PCC 7942, Anabaena 7120, Prochlorococcus sp. (surface and deep water ecotype) Symbiodinium sp., Coscinodiscus wailesii

di and monocots,conifers, brown and red algae, cyanobacteria; cellular [compartment marker] of thylakoid membrane

no confirmed exceptions from predicted reactivity known in the moment

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

Rodríguez-Herva et al. (2012). A bacterial cysteine protease effector protein interferes with photosynthesis to suppress plant innate immune responses. Cell Microbiol (ahead of print)

Petrou et al. (2010). Rapid photoprotection in sea-ice diatoms from the East Antarctic pack ice- Limmol Oceanogr. 3: 1400-1407. (PsbA quantitation in diatoms)

Fristedt et al. (2009). Phosphorylation of photosystem II controls functional macroscopic folding of photosynthetic membranes in Arabidopsis. Plant Cell 21:3950-3964

Brown et al (2007) Resource dynamics during infection of Micromonas pusilla by virus MpV-Sp1. Environmental Microbiol 11: 2720-2727

application example

2 µg of total protein from (1) Arabidopsis thaliana leaf extracted with Protein Extration Buffer, PEB (AS08 300), (2) Hordeum vulgare leaf extracted with PEB, (3) Chlamydomonas reinhardtii total cell extracted with PEB, (4) Synechococcus sp. 7942 total cell extracted with PEB, (5) Anabaena sp. total cell extracted with PEB were separated on  4-12% NuPage (Invitrogen) LDS-PAGE and blotted 1h to PVDF. Blots were blocked immediately following transfer in 2% ECL Advance blocking reagent (GE Healthcare) in 20 mM Tris, 137 mM sodium chloride pH 7.6 with 0.1% (v/v) Tween-20 (TBS-T) 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-rabbit IgG horse radish peroxidase conjugated recommended secondary antibody AS09 602) diluted to 1:50 000 in 2% ECL Advance blocking solution for 1h at room temperature with agitation. The blots were washed as above and developed for 5 min with ECL Advance 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).