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Immunolocalization of various CKXs in maize

Immunolocalization of various CKXs in maize. David Zalabák. Cytokinin Meeting, Olomouc – September 8, 2011. Introduction. Werner et al. 2003 Šmehilová et al. 2009 Subcellular localization of maize CKXs using direct immuno-techniques and TEM. Aims. Heterologous expression of ZmCKXs

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Immunolocalization of various CKXs in maize

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  1. Immunolocalization of various CKXs in maize David Zalabák Cytokinin Meeting, Olomouc – September 8, 2011

  2. Introduction • Werner et al. 2003 • Šmehilová et al. 2009 Subcellular localization of maize CKXs using direct immuno-techniques and TEM.

  3. Aims • Heterologous expression of ZmCKXs • E. coli; pTYB12; intein tag • 2; 3; (4a); 4b; (5); 10 and 12 • Biochemical characterization • When all CKX purified

  4. 2 3 10 2 3 10 2 2 3 3 10 10 2 3 10 Aims • Rabbit polyclonal antibodies • anti-whole protein ZmCKX2 and ZmCKX10 Anti-ZmCKX10 (loaded) Y unspecific IgGs (bound) anti-ZmCKX2 Ab Purif.on ZmCKX10 anti-ZmCKX2 Ab Purif.on protein A Y ZmCKX2 matrix Y Y Y Y anti-ZmCKX10 Ab Purif.na proteinu A ZmCKX10 specific IgGs (in flow through) anti-ZmCKX10 Ab Purif. on ZmCKX2

  5. Aims • Rabbit polyclonal antibodies • anti-peptide ZmCKX4b and ZmCKX10 • (anti-ZmCKX1 and anti-ZmCKX6 in progress) • unique surface 12-14 aa peptide • GenScript • Specific on recombinant ZmCKXs • Testing on protein extracts • co-IP and MS identification of precipitating proteins

  6. Aims • Transmission Electron Microscopy (TEM) • optimized protocol for specimen processing • root tips and embryo tissue • ultramicrotomy (70 nm sections) • immuno-staining • contrast staining (Uranyl acetate)

  7. Ultrastructure of cells in root tip of maize.

  8. Ultrastructure of cells in root tip of maize.

  9. ZmCKX2 localization in cells of root tip of maize.

  10. ZmCKX2 localization in cells of root tip of maize.

  11. ZmCKX2 localization in cells of root tip of maize.

  12. Thank you!

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