Capillary fluorescent electrophoresis (CAFÉ) analysis was performed according to methods described previously by Piparia et al. and Brunn et al. (49 (link), 50 (link)). Tissue lysates and plasma samples were diluted in sample buffer (HT Pico protein express kit; Perkin-Elmer, Waltham, MA) with 10 nM N-ethylmaleimide (Chem-Impex, Inc., Wood Dale, IL) and added to 96-well skirted PCR plates (Thermo-Fisher, Waltham, MA). Dilutions were heated by using a thermal cycler (C1000 Touch; Bio-Rad, Hercules, CA) for a single 10-min cycle at 80°C and run on a LabChip GXII instrument with an HT Pico protein express chip (Perkin-Elmer, Waltham, MA). Peak area analysis was performed using WinNonlin (Pharsight, Mountain View, CA), and the total fluorescent peak area under the curve (AUC) within pertinent molecular weight ranges (2 to 16, 16 to 24, and 24 to 120 kDa) was determined and graphed using Prism software (GraphPad).
Labchip gxii instrument
Manufactured by PerkinElmer
Sourced in United States
The LabChip GXII is a microfluidic-based automated electrophoresis system designed for the rapid analysis of proteins, DNA, and RNA samples. It provides high-resolution separation and quantification of analytes in a sensitive, reproducible, and automated manner.
Automatically generated - may contain errors
Lab products found in correlation
C1000 touch, by Bio-Rad (1 mentions)
96 well skirted pcr plates, by Thermo Fisher Scientific (1 mentions)
N ethylmaleimide, by Chem-Impex (1 mentions)
Prism software, by GraphPad (1 mentions)
Winnonlin, by Pharsight (1 mentions)
Miseq instrument, by Illumina (1 mentions)
Dneasy blood tissue kit, by Qiagen (1 mentions)
Ampure xp bead, by Beckman Coulter (1 mentions)
Nextera xt dna sample preparation kit, by Illumina (1 mentions)
3 protocols using labchip gxii instrument
1
Capillary Fluorescent Electrophoresis for Protein Analysis
2
High-throughput Breastmilk Protein Analysis
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The following major BM proteins were measured in all 450 BM samples: α-lactalbumin, serum albumin, lactoferrin and all caseins. Due to the large number of samples, a classical approach using, for example, gel electrophoresis or HPLC separation did not provide sufficient throughput and speed. Therefore, an innovative microfluidic chip based quantitative method was specifically implemented and validated for BM protein analysis. The method was established on a LabChip GX-II instrument (Perkin Elmer, Waltham, MA, USA) allowing high-throughput analysis in a 96-well format. The principle of this technique is based on traditional SDS-PAGE protein separation but the whole procedure (separation, staining and detection) is integrated and fully automated in a microfluidic system. Results are provided in digital format (no gel staining or scanning, etc.). The general approach of this method was described previously [30 (link)] for bovine milk protein analysis and needed some slight adaptations for the BM sample analysis as described below.
3
Illumina MiSeq Library Preparation Protocol
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Genomic DNA was extracted using a DNeasy® blood & tissue kit (Qiagen, Germany) according to the manufacturer’s instructions. Preparation of the MiSeq library was performed using an Illumina Nextera XT DNA sample preparation kit (Illumina, USA) with minor modifications. In brief, 1 ng of genomic DNA was fragmented in 5 µL of Amplicon Tagment Mix and 10 µL of Tagment DNA buffer. Tagmentation reaction was performed by incubation at 55°C for 5 minutes followed by neutralization with 5 µL of Neutralise Tagment Buffer for 5 minutes. Tagmented DNA (25 µL) was indexed in a 50-µL limited-cycle PCR (12 cycles) as outlined in the Nextera XT protocol and subsequently purified using 25 µL of AMPure XP beads (Beckman Coulter, Australia). The fragment-size distribution of the purified DNA was analysed by the Australian Genome Research Facility utilizing the PerkinElmer LabChip GXII instrument. DNA libraries were adjusted to 2 nmol/L, pooled in equal volumes and then denatured with 0.2 N NaOH. The libraries were sequenced using the 2 × 250 paired-end protocol (MiSeq Reagent Kit v2 for 500 cycles) on an Illumina MiSeq instrument.
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