Electrophoresis and the Agilent Bioanalyzer

Advanced Biotechnology Lab I

Florida Atlantic University

January 23, 2008

Introduction

Electrophoresis is one of the most commonly-used 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.

Theory

The velocity of a charged particle through an electrophoretic medium is given by:

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.

v=q⋅Ef

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.

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

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.

Capillary electrophoresis (cont.)

The output of a CE separation is an electropherogram. For a gel-filled capillary, the smallest molecules will appear first.

Agilent Bioanalyzer

The Agilent Bioanalyzer is based on microfluidics technology and is also capable of performing electrophoretic separations.

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.

Welcome to our e-seminar:

Protein Characterization using theAgilent 2100 Bioanalyzer

Slide 5

The Future of Gel Electrophoresis using Lab-on-a-Chip Technology

DNA 7500 LabChip® kitSizing and Quantitation (100-7500bp)Restriction digests, PCR products

DNA 12000 LabChip® kitSizing and Quantitation (100-12000bp)Restriction digests, larger DNA fragments

RNA 6000 Nano LabChip® kitQuality/Integrity Assessment & Quantitation(up to 6000 bases)Total RNA, mRNA, microarray samples

DNA 1000 LabChip® kitSizing and Quantitation (25-1000bp)Small PCR products, small restriction digests

Protein 200 Plus LabChip® kitSizing and Analysis (14-200 kD) Proteins

DNA 500 LabChip® kitSizing and Quantitation (25-500bp)PCR products, restriction digests

The Agilent 2100 Bioanalyzer

Cell Fluorescence LabChip® kitAnalysis of cell fluorescence parameters Cells

Slide 2Smaller - Faster - Smarter

Scale• small sample volumes• reduced reagent usage• less waste• reduced bench space

00:00:1500:00:1500:00:1500:00:1500:00:1500:00:1501:30:00

Automation• reduced hands-on time • improved accuracy• improved precision• increased productivity

Speed• short analysis times

Lab-on-a-Chip - General Features and Benefits

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Glass or Quartz

MaskLight

2. Develop

3. Etch

4. Bond1. Expose

Etched Channel Plate

Bonded Chip

Glass or Quartz

5. Dice chips6. Mount onto caddy

Courtesy of Caliper Technologies Corp.,Mountain View, CA, USA

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Courtesy of Caliper Technologies Corp.,Mountain View, CA, USA

Slide 4

Lab-on-a-Chip - Principle of Injection & Separation

3

2

4

Direction of electrodriven movement of sample

1

Separation channel

Slide 11

destain solution

gel/dye mix

sample

ladder

chip priming

separation

destaining

detection

Protein 200 Plus LabChip - Chip Layout

Slide 12

Protein Chip - Staining and Detection withIntercalating Fluorescent Dye

protein

micelles

destainSDS + dye

detection

detection

low backgroundgood signal to noise ratioSDS conc. below CMC

high backgroundbad signal to noise ratioSDS conc. above CMC

Slide 27

Prep Chip

15 min

Run Gel

30 min-2 hours

Prep Gel

15 min

40 min

2 hours- 1 day

Scan / Analyze

30 min

Stain / Destain

30 min-8 hours

Run Chip Analyzed - Archived data

25 min

LabChip or Gel - A Faster Answer !

Slide 28

Comparison of SDS-PAGE and Bioanalyzer forProtein Analysis SDS-PAGE Bioanalyzer

Detection Indirect (based on dye binding) Indirect (based on dye binding)

Technology Acceptance Current industry standard New technology

Sample throughput 10-20 samples per gel 10 samples per chip

Waste generation 1.5 l < 0.003 l

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

Capital Cost $ 8 - 15,000(incl. imaging equipment) $ 20,000

Consumable cost (per sample) $ 1 - 2 $ 2

Automation none Separation,staining/destaining, detection

Slide 8

Protein 200 Plus LabChip Kit - Specifications

• automated analysis of 10 protein samples in less than 30minutes

• single assay capable of analyzing proteins ranging in size from14-200 kDa

• sizing resolution of 10% across the size range • large linear dynamic range

(e.g. from 20 - 2000 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 9

12 µ lgel/dye mix

6 µl ladder(denatured)

6 µl sample(denatured)

12 µl destainsolution

Protein Chip Preparation 1. Prepare Chip

Slide 10

Agilent 2100 Bioanalyzer

16 pin electrodesconnected toHV-sources

Exchangeable cartridgefor different assays

Chip holder withheater plate

Optics for detection

2. Run Analysis

Slide 13

Agilent 2100 Bioanalyzer Software

Size and concentrationin digital format

PressSTART

Comparisonof multiplesamples inoverlay mode

Real-time datadisplayed asgel-like image andelectropherogram

File information

Assayinformation

Statusinformation

3. See Data