Peanut (Combrasil - 13L02MCN0049) protein extraction was performed as described by Landry [8], and adapted by Teixeira [12]. In short, seeds were milled in an electric coffee grinder (Philco PERFECT COFFEE 127V model 53901040) and passed through a fine stainless-steel sieve. Samples of peanut flour were placed in 4 Falcon tubes. To each of the tubes containing the seed flours we added one of the buffers to be tested at a final ratio of 1:10 w/v. Namely borate buffer (BB), borate buffer with addition of 2% β-mercaptoethanol (Sigma-Aldrich, São Paulo, Brazil, M3701) (BB-2βME), saline buffer (SB) or Tris/HCl buffer (TB/HCl). All 4 tubes were then placed on a rocker at room temperature for an hour. The material of each tube was paper-filtered, and the eluted solution (crude extract) was centrifuged three times at 5°C and 28,000 × g for 15 minutes (IEC – International Centrifuge, model PR-2, Needham, MA, USA). At the end of each centrifugation, the top layer (containing fat) and the seed precipitate were discarded. The intermediate supernatants were collected, recentrifuged or aliquoted and stored at -20°C until use. Thus, we obtained four crude peanut extracts: crude peanut extract - Borate Buffer (CPE-BB), crude peanut extract - Borate Buffer + 2% β-mercaptoethanol (CPE-BB2βME), crude peanut extract - Saline Buffer (CPE-SB), Crude Peanut Extract - Tris/HCl Buffer (CPE-TB/HCl).

Protein quantification of the crude extracts was determined by the Lowry technique [11]. Protein profile was analyzed using 15% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) as described by Laemmli [13] and stained with Coomassie Brilliant Blue (Bio-Rad, Rockville Centre, NY 11571) [14]. Immunoblotting, to study the immunoreactivity of the proteins was performed as described by Towbin et al [15].