Abstract
The plastid encoded polymerase (PEP) represents a major transcription machinery in mature chloroplasts (Liere et al., 2011; Zhelyazkova et al., 2012). The proper assembly of this multi-subunit complex is important for plant growth and development (Pfalz and Pfannschmidt, 2013). The PEP polymerase can be purified from soluble and from membrane-bound (also named transcriptionally active chromosome, TAC) fractions. Blue Native polyacrylamide gel electrophoresis (BN-PAGE) and sucrose gradient sedimentation followed by immunoblot analyses is used to detect the status of the PEP complex assembly.
Keywords: Enrichment of the plastid encoded Polymerase (PEP), RNA Polymerase assembly, Blue native PAGE (BN-PAGE), Sucrose gradient centrifugation, Plastid protein complexes
Materials and Reagents
Equipment
Procedure
Representative data
Figure 2. Separation of protein complexes by BN-PAGE and subsequent analyses of PEP complex assembly by immunoblotting with antibodies directed against the subunits RpoA and pTAC12. Approximately 50 µg of total leaf protein from the base section of second leaves were loaded per lane. RpoA, RNA polymerase alpha subunit; pTAC12, plastid Transcriptionally Active Chromosome 12 (Note 2). Figure 3. Sucrose-gradient sedimentation. Total leaf proteins from basal half of second leaf were solubilized in BN-Lysis buffer with sonication and soluble fractions subjected to sucrose gradient centrifugation for analysis of PEP-complex assembly (Pfalz et al., 2014). Fractions were collected and immunoblotted with antibodies against pTAC12, RpoA and Rubisco (RbcL). Respective fractions containing Rubisco, PEP (plastid-encoded RNA polymerase), and TAC (transcriptionally active chromosome) (Note 2) are indicated by bars.
Notes
Recipes
Note: All buffers should be stored at 4 °C prior use unless otherwise noted.
Acknowledgments
The work was supported by the DFG (PF667-4). This work is adapted from the work previously published (Pfalz et al., 2015). BN-PAGE is performed essentially as described by Schägger and von Jagow.
References
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