2D‐isolation of pure plasma and thylakoid membranes from the cyanobacterium Synechocystis sp. PCC 6803

Abstract

Aqueous polymer two-phase partitioning in combination with sucrose density centrifugation offered, for the first time, a 2D-separation method for the isolation of pure plasma and thylakoid membranes from the cyanobacterium Synechocystis 6803 without any cross-contaminations. The purity of the membrane fractions was verified by immunoblot analysis using antibodies against membrane-specific marker proteins. As an initiation of a proteomics project, two prominent proteins, which were observed only in the plasma membrane (Slr1513, a hypothetical protein, and HofG, a general secretion pathway protein), or in the thylakoid membrane (PsaE, a photosystem I protein, and NdhH, a subunit of NADH dehydrogenase), were identified.z 1998 Federation of European Biochemical Societies.

Operon flv4-flv2 Provides Cyanobacterial Photosystem II with Flexibility of Electron Transfer

“…Unexpectedly, only a weak response was detected with Flv4 and Flv2 antibodies compared with that recorded in the total membrane fraction isolated by a standard method used in our laboratory (which includes high cation concentration; see Methods). However, no Sll0218 signal could be found in conventional two-phase fractions (Norling et al, 1998;Figure 2A).…”

“…Although no transmembrane helices were predicted in any of the FDP sequences, we also found a strong association of the Flv2 and Flv4 proteins with the membrane fraction . To localize the Flv4, Sll0218, and Flv2 proteins precisely in the membrane fraction, the thylakoid and plasma membranes, purified by the two-phase partitioning method (Norling et al, 1998), were probed with protein specific antibodies. Unexpectedly, only a weak response was detected with Flv4 and Flv2 antibodies compared with that recorded in the total membrane fraction isolated by a standard method used in our laboratory (which includes high cation concentration; see Methods).…”

“…Nearly complete absence of the Flv4, Sll0218, and Flv2 proteins in the thylakoid and plasma membrane fractions purified by the conventional two-phase partitioning system (Norling et al, 1998) (Figure 2A) needed clarification. To this end, the Flv4, Sll0218, and Flv2 proteins were first analyzed from the total membrane (thylakoids plus plasma membrane) and soluble fractions of the wild-type cells.…”

New insights into iron acquisition by cyanobacteria: an essential role for ExbB-ExbD complex in inorganic iron uptake

“…Cytoplasmic and thylakoid membranes were isolated by the 2D-separation method combined with sucrose density centrifugation (Norling et al, 1998;Huang et al, 2006). Samples were resolved on 12% sodium dodecyl sulfate polyacrylamide gel and immunoblotted with corresponding antibodies.…”

Current trends in high throughput proteomics in cyanobacteria

“…PCC 7120 [13,24]. For example, earlier reports by Norling et al demonstrated the extensive optimisation required to profile and study thylakoid and membrane-bound proteins and their successful characterisation [20,25,26]. Other notable published studies include the early work on Nostoc sp.…”