Page 248 - The Manga Guide to Biochemistry

Prepare the gel so that it Thin membrane,
nitrocellulose membrane,
carries a negative charge ( – ).
or PVDF membrane
(use the reagent called SDS)
Attach gel
Add the protein to membrane
sample. as described
above, and apply
Polyacrylamide gel – electricity to
(thickness: approximately transfer the
1 mm; height: several cm to + proteins in the gel
– to the membrane
several dozen cm)
(blotting).
Pass a current through.
+
Proteins are separated –
according to size.

Smaller proteins
move farther down.

+ Perform experiments on this
membrane using various methods
These marks that appear when (Like Lectin blotting, for example).
proteins are detected are
called protein bands. Western Blot

SDS-polyacrylamide
gel electrophoresis

Lectin Blotting

Lectins are proteins that can bond specifically to certain sugar chains. Because lectins will
bond differently according to the type of sugar chain, lectins can be used to identify the type
of sugar chain that is bonded to a protein. A method similar to a western blot can be used
after proteins are transferred onto a membrane. Various lectins are mixed with the proteins,
and only the lectins that reacted are detected. This lets us identify the types of sugar chains
that exist in the proteins that were transferred onto the membrane. This is called lectin
­blotting.

The following figure shows an experiment in which a lectin called wheat germ agglu-
tinin (WGA) identifies a sugar chain to which the saccharide called N-acetylglucosamine
(GlcNAc) is attached.

In this lectin blot performed for the rough protein fraction of the starfish oocyte, it is
apparent that two large bands are glowing brightly. WGA was the lectin used here.

234 Chapter 5