Protein 80 kit

The molecular weights of the proteins in the fermented and unfermented lentil samples and dairy and soy controls, as well as in the soluble fractions after fermentation, was analysed using an Agilent Bioanalyzer 2100 Lab-on-a-Chip capillary electrophoresis system with an Agilent Protein 80 kit. For the soluble protein fraction, the fermented samples were centrifuged at 4000× g for 10 min, and the supernatant was used directly for the protein profile analysis. Proteins of the fermented and unfermented samples were extracted using the method from [43 (link)], using a protein concentration of 0.5% w/v. DTT was added for reducing conditions. As it has been shown that there are only minor differences between the reducing and non-reducing protein profiles of lentil protein using this method [33 (link),44 (link)], non-reducing conditions have been omitted.

An Agilent Bioanalyzer 2100 Lab-on-a-Chip capillary electrophoresis system was used to analyse the protein profile and estimate the molecular weights of the respective protein bands. Samples were prepared according to Amagliani et al. [31 (link)] with slight modifications: LPIs were dispersed in 2% SDS, 2 M thiourea and 6 M urea, to give a protein concentration of 2.5 mg/mL. Dispersions were shaken for 2 h at 22 °C, and centrifuged to remove insoluble material. Samples were analysed using an Agilent Protein 80 kit and Protein 230 kit according to the instructions within the ranges of 5–80 and 14–230 kDa, respectively. For reducing conditions, DTT was included in the sample buffer according to kit instructions.

Monospecific antibodies and κλ body distribution and integrity were assessed by electrophoresis, isoelectric focusing gel analysis (IEF), hydrophobic interaction high performance liquid chromatography (HIC-HPLC) and size exclusion high performance liquid chromatography SEC-HPLC. Purified IgGs were analyzed by electrophoresis in denaturing and reducing conditions. The Agilent 2100 Bioanalyzer was used with the Protein 80 kit as described by the manufacturer (Agilent Technologies, Santa Clara, CA, USA). The distribution of the different formats of IgG (monospecific lambda, kappa and bispecific antibody) was determined by isoelectric focusing (Cambrex pH 7–11 IsoGel agarose plates) and HIC-HPLC analysis using ProPac HIC-10 column (Dionex, Sunnyvale, CA, USA). A gradient of mobile phase A (0.01 M sodium phosphate dibasic buffer (Sigma-Aldrich, St Louis, MO, USA) + 1.5 M ammonium sulphate (Sigma-Aldrich, St Louis, MO, USA), pH 3.5) from 100 to 10% and a growing gradient of mobile phase B (0.01 M sodium phosphate dibasic buffer + 10% acetonitrile (Merck KGaA, Darmstadt, Germany), pH 3.5) from 0 to 90% were applied. A blank was performed with mobile phase A, pH 7.0. Aggregate and fragment levels were determined by SEC-HPLC with a Biosep-SEC-s3000 column (Phenomenex, Torrance, CA, USA) using a 200 mM sodium phosphate dibasic buffer, pH 7.0 mobile phase.