Immunoblotting Analysis of nsLTP1 in Cereal Grains with Antiserum Raised against Recombinant Rice nsLTP1

Abstract

Nonspecific lipid transfer protein 1 (nsLTP1) is widely distributed in plants and has been recognized as a widely cross-reacting plant pan-allergen. In the present study, a recombinant protein of rice nsLTP1 was prepared with an Escherichia coli expression system, and polyclonal antiserum was raised by immunizing rabbits with this recombinant nsLTP1. The antiserum specifically immunoreacted with rice nsLTP1 and homologous nsLTP1 proteins of other kinds of cereal grains. Moreover, the antiserum was applied to compare nsLTP1 content among rice cultivars and among rice fractions separated by grain-milling. Variation of nsLTP1 contents were observed among rice cultivars, and rice nsLTP1 was proved to be mainly located in the outer layer of rice grains, providing evidence for decreasing nsLTP1 content by grain-milling.

Translocation of Barley β-amylase into Rice Grains during Cooking Rice Mixed with Barley (Mugimeshi)

“…3-2, lanes G-I; open arrowhead). Lang reported that variation in the migration speed of LTP1 was observed among rice, millet, rye, and barley (Lang et al, 2011). Differences in migration speed between barley and rice LTP1 is consistent with Lang's report, which used a rabbit polyclonal antibody against recombinant rice LTP1 (Lang et al, 2011).…”

Evaluation of Extraction Solutions for Biochemical Analyses of the Proteins in Rice Grains

“…The rabbit antisera were prepared by immunizing rabbits with the respective recombinant proteins as previously described. 28,32) Immunoblotting and signal detection were also carried out as previously described. 28) The chemiluminescent signal was detected by the Pharos FX molecular imager system (Bio-Rad Laboratories), the signal intensity being quantified by using Quantity One 1-D Analysis software (Bio-Rad Laboratories).…”