Lab on a chip bioanalyzer 2100

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.

The protein profiles of the samples were analysed regarding their molecular weights using an Agilent Bioanalyzer 2100 Lab-on-a-Chip capillary electrophoresis system with an Agilent Protein 230 kit. The samples were prepared based on Amagliani et al. [43 (link)], with slight modifications. First, protein isolates were dispersed in an extraction buffer of 2% SDS, 2 M thiourea and 6 M urea to reach a protein concentration of 0.5%. After shaking on a platform shaker at room temperature for 2 h, the dispersions were centrifuged (3000× g, 30 min) to remove insoluble material. Dithiothreitol (DTT) was added to the sample buffer according to kit instructions for stronger reducing conditions.

Independently derived 70Z/3 lines infected with MSCV, or MSCV-miR-24-2 were generated previously[65 (link)]. Four MSCV lines and 2 MSCV-miR-24-2 lines were examined. Total RNA was prepared using Trizol reagent (Invitrogen, Carlsbad, CA). RNA quality was evaluated using the Lab-on-a-Chip Bioanalyzer 2100 (Agilent, Palo Alto, CA). Biotinylated cRNA was prepared according to the standard Affymetrix protocol using the Superscript Choice System (Invitrogen) and the RNA transcript labeling kit (ENZO, Farmingdale, NY) for cRNA preparation. Following fragmentation 10ug of cRNA was hybridized to Affymetrix Mouse 430 2.0 Gene Chip. Microarray analysis was performed using the Affymetrix GeneArray Scanner G2500A. The Affymetrix Expression Console Software Version 1.4.0 was used to create summarized expression values (CHP-files) from expression array feature intensities (CEL-files). Raw data were normalized with robust multichip analysis (RMA) with Affymetrix transcriptome analysis console (TAC) software. DNA microarray and sample annotation data were deposited in GEO under the accession number GSE65874.