Monoclonal antibodies used for the characterization of the two putative iron-sulphur centre proteins associated with photosystem I

Abstract

Monoclonal antibodies have been produced to the two putative 18.3 and 15.2 kD iron-sulphur centre proteins in barley. When photosystem I particles, containing only chlorophyll a-protein 1 and the 18.3 and 15.2 kD proteins, were used as the antigen, thirteen independent clones secreting either IgG or IgM antibodies against the 15.2 kD protein were formed. No hybridomas secreting antibodies to the 18.3 kD protein were detected. When a delipidated polypeptide preparation was the immunogen, we obtained one hybridoma, secreting antibodies of the IgM class to the 18.3 kD protein.These antibodies were used in immune-blot assays. Neither the 18.3 nor 15.2 kD putative iron-sulphur centre proteins were detected in etioplast membranes. However, in the chloroplast membranes of the photosystem I mutant viridis-zb ~' we found small amounts of these proteins.The two proteins were purified by electroelution and then tested by immune-blot assay. Fractions with positive reaction were collected and the amino acid compositions determined. There were significant differences in the amounts ofserine, glycine and histidine between the two proteins.

Probing in vitro translation products with monoclonal antibodies to a 15.2 kD polypeptide subunit of photosystem I

“…Monoclonal antibodies have been prepared to the three Coomassie stainable polypeptide subunits of the barley PSI particle (15, 17,18,21), with the aim of determining their sites of synthesis. We obtained both IgG and IgM secreting hybridomas to the chlorophyll a-protein 1 and the 15.2 kD protein.…”

“…It was initially thought that IgMs were suitable for immunoprecipitations by introducing,a secondary antibody of the IgG class. Rabbit antimouse immunoglobulins reacted well with the monoclonal IgM's in immune-blot assays ( 15, 16,17,21), but immunoprecipitation with the monoclonal IgM's was not successful (Figure 4). It was thus not possible for us to determine the site of synthesis of the 18.3 kD PSI polypeptide (17).…”

“…Immediately after completion of the electrophoretic run the polypeptides were transferred from the SDS-gel to nitrocellulose filters (18,21,40). These were used for immune-blot assays, exactly as described previously (21).…”

“…Bands were visualized using peroxidase coupled rabbit antimouse immunoglobulins (DAKO Immunoglobulins A/S P 161). (21), using PSI particles as the antigen. The amount of monoclonal antibody used was estimated by multiplying the hybridoma cell concentration by 0.05 ng, which is the amount of antibody that one hybridoma cell secretes during 48 hours (38).…”

Probing in vitro translation products with monoclonal antibodies to chlorophylla/b-binding proteins of barley thylakoids

“…Immediately after completion of the electrophoretic run, the polypeptides were transferred from the SDS-gel to nitrocellulose filters (10,12,24). These were used for immunoblot assays as previously described (12).…”