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!