Electrophoresis and the Agilent Bioanalyzer. Advanced Biotechnology Lab I Florida Atlantic University January 23, 2008
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1 Electrophoresis and the Agilent Bioanalyzer Advanced Biotechnology Lab I Florida Atlantic University January 23, 2008
2 Introduction Electrophoresis is one of the most commonlyused methods of separating proteins and nucleic acids. phoresis, from the Greek, means a carrying Electrophoresis is literally the carrying of a charged particle by an electric field A viscous medium (gel) is usually used to retard the movement of the particle The separation can be carried out in a slab gel, a tube or a microfluidic channel.
3 Theory The velocity of a charged particle through an electrophoretic medium is given by: v= q E f where q is the charge on the particle, E is the electric field strength and f is the frictional coefficient between the particle and the separation medium. We can see how larger particles, which have a higher frictional coefficient, will migrate through the medium at a slower rate. Thus, we can separate particles by size.
4 SDS-PAGE The ionic detergent SDS is used to denature proteins and create particles with an equal charge-to-mass ratio. An electric field is then used to separate the proteins. Either Coomassie blue or silver can be used to stain the gel.
5 Capillary electrophoresis (CE) Employs capillary tubing within which the electrophoretic separation occurs Utilizes very high electric field strengths for fast separations Uses modern detector technology which provides a computer-generated electropherogram Requires minute amounts of sample and reagents Is easily automated for precise quantitative analysis and ease of use
6 Capillary electrophoresis (cont.) After filling the capillary with a polymer gel and injecting a sample, separation takes place under a high-voltage electric field. Analytes are monitored and recorded as they pass the detector.
7 Capillary electrophoresis (cont.) The output of a CE separation is an electropherogram. For a gelfilled capillary, the smallest molecules will appear first.
8 Agilent Bioanalyzer The Agilent Bioanalyzer is based on microfluidics technology and is also capable of performing electrophoretic separations.
9 Agilent Bioanalyzer (cont.) The Lab-On-A-Chip has tiny channels analogous to the capillary tubing from CE. Proteins or other analytes are subjected to an electric field, migrate along a separation channel and are picked up by the detector.
10 Welcome to our e-seminar: Protein Characterization using the Agilent 2100 Bioanalyzer
11 The Agilent 2100 Bioanalyzer The Future of Gel Electrophoresis using Lab-on-a-Chip Technology RNA 6000 Nano LabChip kit Quality/Integrity Assessment & Quantitation (up to 6000 bases) Total RNA, mrna, microarray samples DNA 500 LabChip kit Sizing and Quantitation (25-500bp) PCR products, restriction digests DNA 1000 LabChip kit Sizing and Quantitation ( bp) Small PCR products, small restriction digests DNA 7500 LabChip kit Sizing and Quantitation ( bp) Restriction digests, PCR products DNA LabChip kit Sizing and Quantitation ( bp) Restriction digests, larger DNA fragments Protein 200 Plus LabChip kit Sizing and Analysis ( kd) Proteins Cell Fluorescence LabChip kit Analysis of cell fluorescence parameters Cells Slide 5
12 Lab-on-a-Chip - General Features and Benefits Scale small sample volumes reduced reagent usage less waste reduced bench space Speed short analysis times 01:30:00 00:00:15 00:00:15 Automation reduced hands-on time improved accuracy improved precision increased productivity Smaller - Faster - Smarter Slide 2
13 7KH/DE&KLS $SSURDFK 'LVSRVDEOHPLFURIDEULFDWHGJODVVFKLS 0DGHRIWZRJODVVOD\HUVRQHZLWK PLFURFKDQQHOVRQHZLWKWKURXJK KROHVERQGHGWRJHWKHU *OXHGLQWRDSODVWLFFDGG\ZKLFK DFFRPPRGDWHVZHOOVIRUJHOVDPSOH EXIIHUHWF +DQGOLQJRIQDQROLWHUDPRXQWVRI OLTXLGV 6OLGHÃ
14 +RZ&KLSVDUH0DGH 1. Expose Light Mask 4. Bond Glass or Quartz 2. Develop Glass or Quartz Etched Channel Plate 3. Etch Bonded Chip 5. Dice chips 6. Mount onto caddy Courtesy of Caliper Technologies Corp., Mountain View, CA, USA 6OLGHÃ
15 &KDQQHOVDIWHU3KRWROLWKRJUDSK\DQG(WFKLQJ Courtesy of Caliper Technologies Corp., Mountain View, CA, USA 6OLGHÃ
16 Lab-on-a-Chip - Principle of Injection & Separation 1 2 Separation channel 3 4 Direction of electrodriven movement of sample Slide 4
17 Protein 200 Plus LabChip - Chip Layout sample chip priming gel/dye mix separation detection destain solution ladder destaining Slide 11
18 Protein Chip - Staining and Detection with Intercalating Fluorescent Dye detection high background bad signal to noise ratio SDS conc. above CMC protein SDS + dye destain detection micelles low background good signal to noise ratio SDS conc. below CMC Slide 12
19 LabChip or Gel - A Faster Answer! 15 min 25 min 40 min Prep Chip Run Chip Analyzed - Archived data 2 hours- 1 day 15 min 30 min-2 hours 30 min-8 hours 30 min Prep Gel Run Gel Stain / Destain Scan / Analyze Slide 27
20 Comparison of SDS-PAGE and Bioanalyzer for Protein Analysis SDS-PAGE Bioanalyzer Detection Indirect (based on dye binding) Indirect (based on dye binding) Technology Acceptance Current industry standard New technology Automation Capital Cost none $ 8-15,000 (incl. imaging equipment) Separation, staining/destaining, detection $ 20,000 Consumable cost (per sample) $ 1-2 $ 2 Sample throughput samples per gel 10 samples per chip Pre-run processing 5-10 min 5-10 min Run time 30 min - 2 hours per gel 25 min per chip Post-run processing 1 hour - 1 day (staining, destaining, densitometry) none (automatic integration) 5 min (manual integration) Total turnaround time 1-2 days 45 minutes Waste generation 1.5 l < l Slide 28
21 Protein 200 Plus LabChip Kit - Specifications automated analysis of 10 protein samples in less than 30 minutes single assay capable of analyzing proteins ranging in size from kda sizing resolution of 10% across the size range large linear dynamic range (e.g. from ng/µl BSA in PBS) sensitivity equivalent to non-colloidal Coomassie (R-250) stain relative and absolute quantitation (semi-quantitative) compatible to a variety of sample buffer components Slide 8
22 Protein Chip Preparation 1. Prepare Chip 6 µl sample (denatured) 12 µ l gel/dye mix 12 µl destain solution 6 µl ladder (denatured) Slide 9
23 Agilent 2100 Bioanalyzer 2. Run Analysis Exchangeable cartridge for different assays 16 pin electrodes connected to HV-sources Chip holder with heater plate Optics for detection Slide 10
24 Agilent 2100 Bioanalyzer Software 3. See Data File information Press START Real-time data displayed as gel-like image and electropherogram Comparison of multiple samples in overlay mode Assay information Size and concentration in digital format Status information Slide 13
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