Agilent protein 80 kit

For purity test of colicins, capillary gel electrophoresis (CGE)-on-a-chip analysis was performed on an Agilent 2100 bioanalyzer (Agilent Technologies Deutschland, Waldbronn, Germany) in combination with an Agilent Protein 80 Kit (sizing range: 5–80 kDa, Agilent Technologies Deutschland, Waldbronn, Germany) and 2100 Expert Software (Agilent Technologies Deutschland, Waldbronn, Germany)) [29 (link)]. All reagents and chips were prepared according to the manufacturer’s instructions.
Lyophilized, buffer-containing colicin samples were reconstituted with water to a concentration of 1 mg colicin per mL. Then, 4 µL of each colicin sample and 2 µL of reducing sample buffer were mixed and incubated at 95 °C for 5 min. After adding 84 µL water to each colicin-buffer mix, 6 µL of each sample was loaded onto a chip together with two BSA standard protein samples (reduced and non-reduced) and a Protein 80 ladder. The chip run results were displayed as a gel-like image, electropherograms, and in tabular form. Peak baseline adjusting and peak integration of electropherograms were done automatically and, if necessary, manual adjusting of peak baselines was done on a case-by-case basis.

Lima bean leaf proteins from plants exposed to GMF and NNMF were characterized with the Agilent Protein 80 Kit by using the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA) according to the manufacturer’s instruction. The same concentration of proteins for each sample obtained from the total protein extraction was used to prepare samples, reaching a final volume of 4 µL. The Protein 80 Chip was then analyzed in the 2100 Bioanalyzer, and the data acquisition and analysis were performed by the Agilent 2100 Expert Software (Agilent Technologies, Santa Clara, CA, USA).

Sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE) was used to detect unbound protein on supernatants (SN). After 1 h of incubation in a rotating wheel, samples were centrifuged for 10 min at 10,000× g at room temperature and SN were collected. The same volume of SN for each sample was also loaded on Protein LabChip gel (according to manufacturer’s instruction), and the presence of unbound protein was detected with Agilent Protein 80 kit with Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). The starting amount of protein, as one of the samples, was loaded as the control. The gel contains three internal controls, which allow for the quantitation of the detected protein by Agilent 2100 software (version B.02.10SI764).