Modifications of Wheat Germ Cell-Free System for Functional Proteomics of Plant Membrane Proteins - Plant Proteomics: Methods and Protocols - page 260

Fig. 4 Synthesis of myristoylated AtCBL1. ( a ) Synthesis of myristoylated AtCBL1. AtCBL1 and AtCBL1(G2A)
were synthesized by batch method in the presence of [ 14 C]myristic acid. After the translation reaction,
the reaction mixture was visualized by SDS-PAGE and either CBB staining ( left ) or autoradiography ( right ).
( b ) Accudenz density gradient ultracentrifugation. AtCBL1 and AtCBL1(G2A) were synthesized by the WGCF
system in the presence or the absence of liposomes and myristic acid. The reaction mixture was subjected to
Accudenz density gradient ultracentrifugation. Fractions were collected from the top of the tube and subjected
to SDS-PAGE and autoradiography

As shown in Fig. 4b , AtCBL1 is found at the top of the centrifuge
tube when AtCBL1 is synthesized in the presence of myristic acid
and liposomes. This technique is useful for rough purifi cation of
N -myristoylated proteins.

1. Prepare 30, 35, and 80 % (w/v) Accudenz solution in ultra-
centrifugation buffer.
2. Mix 150 μ l of the translation mixture with 150 μ l of ultracen-

trifugation buffer and 300 μ l of 80 % Accudenz solution.

3. Transfer 600 l of the resultant 40 % Accudenz solution con-

taining the translation mixture to an ultracentrifugation tube.

μ