Immunolocalization of various CKXs in maize David Zalabák Cytokinin Meeting, Olomouc – September...
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Transcript of Immunolocalization of various CKXs in maize David Zalabák Cytokinin Meeting, Olomouc – September...
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• E. coli; pTYB12; intein tag• 2; 3; (4a); 4b; (5); 10 and 12
• Biochemical characterization• When all CKX purified
Aims• Rabbit polyclonal antibodies
• anti-whole protein ZmCKX2 and ZmCKX10
anti-ZmCKX2 Ab
Purif.on protein A
2 3 10
2 3 10
2 3 10
2 3 10
anti-ZmCKX10 Ab
Purif. na proteinu A
anti-ZmCKX2 Ab
Purif. on ZmCKX10
anti-ZmCKX10 Ab
Purif. on ZmCKX2
2 3 10
Zm
CK
X2
mat
rix YY
Yunspecific IgGs
(bound)
ZmCKX10 specific IgGs
(in flow through)
Y
Anti-ZmCKX10
(loaded)
Y Y
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
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)
Ultrastructure of cells in root tip of maize.
Ultrastructure of cells in root tip of maize.
ZmCKX2 localization in cells of root tip of maize.
ZmCKX2 localization in cells of root tip of maize.
ZmCKX2 localization in cells of root tip of maize.
Thank you!