iman kishawi, phd senior application scientist
TRANSCRIPT
Page 1 2008
2100 Bioanalyzer
Iman Kishawi, PhDSenior Application Scientist
Page 2 2008
The Lab-on-a-Chip Approach
Sample volumes 1 - 5 µl
10 -12 samples depending on Assay
Separation, staining, detection of samples
Results in 5-30 minutes available
No extra waste removal needed
Disposable Chip, no crosscontamination
Increasing quality and speed of gel electrophoresis
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Agilent 2100 Bioanalyzer
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2. Run Analysis
Page 5 2008Page 5
Bioanalyzer Overview
November 2007
Current 2100 Analysis Kits
DNA Assays:1000, 7500, 12000• Sizing
• Quantitation
• PCR products, digests,
larger DNA fragments
• 12 samples in 30 min.
Cell Assays:Flexible use
• Analysis of 6 samples • Two color detection• Analysis of protein expression
in cells
Protein Assays:P80, P230, HSP-250• Sizing
• Quantitation
• cell lysates, column fractions,
purified proteins, antibodies etc.
• 10 samples in 40 min.
RNA Assays:nano, pico, Small RNA
• Quantitation (Sizing in Small RNA)
• total RNA, mRNA
• purity & integrity determination
• 10 samples in 30 min.
Electrophoretic Separations
Flow Cytometry
Page 6 2008
Cell Applications
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Video
Page 7 2008
Cell Assay
Apoptosis
Transfection Efficiency Monitoring
• Detection of GFP-transfected cells• Antibody staining: Detection of transfected cells expressing the
encoded protein
Protein Expression Monitoring
• Extracellular and Intracellular Antibody staining for detection of protein expressed on the cell surface, in the cytoplasm, or in the nucleus
Gene silencing
Page 8 2008
Principle of Pressure-Driven FlowFor cell assays (analysis of cell fluorescence parameters)On-chip simple flow cytometric studies
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Page 9 2008
The Bioanalyzer Lab-on-a-Chip Approach
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Flow cytometry on the chip - Detection Animation
Page 11 2008
Flow Cytometry on a Chip - Hydrodynamic Focusing
All six cell samples arehydrodynamicly focused to one side of the micro channel
At each of the six pinch points the cell stream is joined by a buffer stream from one of the two buffer wells
The two liquids do not mix immediately
The cells then move towards the detector in single file
Page 12 2008
Flow Cytometry on a Chip - Two Color Detection- Three Types of Events
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Page 13 2008
Some Target applications
Apoptosis:Annexin V Detection of phosphatidylserine on the cell surfaceCaspase-3 Detection of activated caspase-3 in the cytoplasm
Transfection Efficiency Monitoring:GFP: Detection of GFP-transfected cellsAntibody staining: Detection of transfected cells expressing the encoded
proteinProtein Expression Monitoring:
Extracellular and Intracellular Antibody staining for detection of protein expressed on the cell surface, in the cytoplasm, or in the nucleus
Gene silencing:Optimization of siRNA transfection procedureVerify silencing by cellular protein expression measurementCorrelation of siRNA uptake and gene knockdown
Page 14 2008
Typical workflow:
Cell assays: sample preparation
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Page 15 2008
2100 Bioanalyzer
Red detection channel:
• 620-645 nm excitation with Laser (Maximum 630 nm)
• 674-696 nm detection range (Maximum 680 nm)
Blue detection channel:
• 458-482 nm excitation with LED (Maximum 470 nm)
• 510-540 nm detection range (Maximum 525 nm)
Flow Cytometry on a Chip - Optics & Detection
Page 16 2008
Cell Assays - Applications: ApoptosisAnnexin Binding
PhosphatidylPhosphatidyl--serine from inner leaflet flips to outer membrane serine from inner leaflet flips to outer membrane during apoptosis and can be labeled by Annexin Vduring apoptosis and can be labeled by Annexin V
Healthy cellHealthy cell
““LiveLive”” apoptotic cellapoptotic cell
Dead cellDead cell
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Page 17 2008
Annexin V Assay (24h Induction)
0%
20%
40%
60%
80%
0 10 20 30
Sample Number
% A
popt
otic 2100-1
2100-22100-3Flow cytometer
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Page 18 2008
Applications: Protein Expression AnalysisGFP Transfection Efficiency Control
CHO-K1 cells were transfected with EGFP DNA and Lipofectamine.
GFP transfected cells
Mock transfected cells
0.1 %
56.6 %
Control
EGFP transfected
Page 19 2008
GFP Transfection Efficiency
0%
10%
20%
30%
40%
50%
60%
70%
1 2 3 4 5
Chip Number
Tran
sfec
tion
Eff
icie
ncy
Flow cyt.
2100-12100-22100-3
2100-1 2100-2 2100-3 All Flow cyt.ctrl mean 0.46 0.31 0.47 0.40 0.16
SD 0.08 0.29 0.43 0.29 0.12GFP mean 60.19 59.15 59.26 59.53 60.90
SD 2.13 2.48 2.10 2.26 1.22%CV 3.54 4.19 3.54 3.80 2.01
Page 20 2008
Flow Cytometry Assays Applications - Cell surface Antibody staining
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Page 21 2008
Extracellular Antibody Staining
Averaged data per instrument
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1 2 3 4 5 6
sample #
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ated
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Flow cytometer
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Mean % CD3+ cells2100-1 2100-2 2100-3 2100-4 Flow cyt.60.9 67.8 66.6 65.0 60.934.4 36.7 36.7 34.3 29.817.3 17.6 18.7 17.2 13.88.9 9.4 9.9 8.3 6.55.1 4.4 5.3 4.9 3.20.8 0.6 0.3 0.3 0.0
Page 22 2008
15 min
cells + CBNF
15 minOn-���
Spin, aspirate,resuspend,spin, aspirate
CBNF
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Resuspend cells in CB
GFP On-Chip Staining - Workflow
Page 23 2008
GFP On-Chip Staining - Histogram Quality
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Lab-on-a-Chip - Principle of Injection & Separation
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The LabChip® Approach - Simplified Model(see chip animation.ppt)
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Page 26 2008
DNA Applications
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Page 27 2008
Application Areas for the DNA Assays
PCR product purity
Multiplex PCR Applications
Gene expression analysis via RT-PCR (target validation)
GMO testing
Pathogen detection (homeland defense, hospitals, environmental)
Genotyping applications
• Duplications/ deletions• Allele frequency• Bacterial sub-typing• Forensics
Cancer diagnostics
Page 28 2008
DNA Kit Specification
Page 29 2008
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Data Format - Gel-Like Image c/w Agarose Gel
Page 30 2008
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300 bp
3000 bp
Determination of PCR Product Impurity
Page 31 2008
GMO Detection: Determination of GM Soya Percentage
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Page 32 2008
Optimization of Multiplex PCR on a 19-plex PCR
Data kindly provided by QIAGEN GmbH, Germany
Page 33 2008
Tumor Diagnostics
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Page 34 2008
Detection of Single Base Mutations (1)��������6�����=��"�������&����.7�;����!��A+��>����� ���� ���� �� �6.88�3��
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Page 35 2008
Detection of Single Base Mutations (2)
wt 106
200 bp109 bp
91 bp
91 bp91+83 bp
168 bp
wt 59
276 bp276 bp
251 bp
1* 2 3 4 5*0
5
10
15
20
25
30
Fluo
resc
ence
Time (seconds)
30 35 40 45 50 55 60 65 70 75
p53Exon 7 wt/HpaII p53Exon 7 clone 59/HpaII
Lower Marker
Upper Marker267/268 bp
251 bp
166 bp
84/85 bp
1* 2 3 4*0
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10
15
20
25
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p53Exon 8 wt/HpaII p53Exon 8 clone 106/HpaII
Lower Marker
Upper Marker
Time (seconds)
208 bp111 bp
90 bp
Page 36 2008
Label free Analysis of Microsatellite InstabilitiesClinical Diagnostics and Molecular Diagnostics of Cancer
Microsatellite instabilities present in 10-15% of colon and gastric carcinomas
Study: 40 cases of colon carcinoma
5 microsatellite loci investigated
Results compared with traditional PAGE:
95% concordance rate
Page 37 2008
RNA Applications
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Page 38 2008
Agilent 2100 bioanalyzer:the industry standard in RNA QC
First commercially available Lab-on-a-Chip product (since October 1999)
Analysis of totalRNA, mRNA and small RNA samples in ng and pg concentration range
Standardized RNA integrity assessment with RIN* algorithm
Multi-analysis capabilities: DNA, RNA, Proteins and Flow Cytometry
January 2008: ~ 7200 citations
Electrophoretic sizing, quantitation and QC of XNA and Proteins on a small glas Chip as done traditionally on slab gels (Agarose or SDS-PAGE)
Number of BioA publications/month
RIN = RNA Integrity Number, an Agilent patented algorithm to Determine RNA quality in a normalized way
Page 39 2008
RNA Kit Specifications
Page 40 2008
total RNA
determine integrity and quality of total RNA
determination of RNA concentration
identify ribosomal peaks
calculate the ratio of ribosomal peaks (18S/28S or 16S/23S)
RNA integrity number (RIN)
mRNA
determine integrity and quality of mRNA samples
Determination of mRNA concentration
calculate % ribosomal RNA in mRNA samples
Features of the RNA 6000 Assays
Problem Description
The ratio of ribosomal bands is not sufficient to describe RNA integrity!
RNA degradation is a gradual process.
Results have to be interpreted by visual inspection.
Overlay of electropherograms only works well for samples with the same concentration.
Instrument dependency in signal height
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Page 42 2008
High quality total RNA
Partially degraded total RNA
18S
28S
Fluo
resc
ence
Time (seconds)
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28S
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resc
ence
Time (seconds)
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()�*)�Typical first QC step after RNA sample prep prior to microarrays or real-time PCR
RNA Quality Control:Assessing Total RNA Integrity
RIN 9.2
RIN 5.8
Page 43 2008
Gel Chip Comparison
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Page 44 2008
RNA QC in Routine Gene Expression Workflow
Start again with sample isolation
Cells / Culture
RNA isolation
Total RNA
RIN
RNA QC via Agilent 2100 bioanalyzer
RIN above threshold
Continue with downstream Experiment (Microarray, real-time PCR, etc.)
Page 45 2008
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cRNA Hybridization - Workflow
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Labeled cRNA Quality Check by NanoDrop
Incorporation of Cy Dye
Yield of cRNAIntact Partial
DegradationCompleteDegradation
0
5
10
15
Yie
ld o
f cR
NA
(ug)
Cy3 Cy5 Cy3 Cy5 Cy3 Cy5
9.2 8.57.4 7.3
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0
5
10
15
Cy3 Cy5 Cy3 Cy5 Cy3 Cy5
Inco
rpor
atio
n of
cR
NA
(pm
ol/u
g)
Intact PartialDegradation
CompleteDegradation
11.312.7
8.110.0
3.04.1
Labeled cRNA Quality Check by BioAnalyzer
- Ladder- Intact (RIN = 7.3)- Partial Degradation (RIN = 5.2)- Complete Degradation (RIN = 2.4)
0.2K
0.5K
1K
2K
4K
6K
Cy3 labeled cRNA
0.2K
0.5K
1K
2K
4K
6K
Cy5 labeled cRNA
Peak Size : 0.6K (Intact)Peak Size : 0.3K (Partial Deg.)
Peak Size : 0.1K (Complete Deg.)
Partial Degradation
RIN = 5.2
Complete Degradation
RIN = 2.4
Intact
RIN = 7.3
- Intact (RIN = 7.3)- Partial Degradation (RIN = 5.2)- Complete Degradation (RIN = 2.4)
Scatter Plots : Self vs. Self
Scatter Plots : Intact vs. DegradationP
artia
l Deg
rada
tion
Intact
RIN = 5.2
RIN = 7.3
Intact vs. Partial Degradation
- Intact (RIN = 7.3)- Partial Degradation (RIN = 5.2)- Complete Degradation (RIN = 2.4)
Com
plet
e D
egra
datio
n
Intact
RIN = 2.4
RIN = 7.3
Intact vs. Complete Degradation
Intact (self to self)
Intact
Inta
ct RIN = 7.3
RIN = 7.3
Conclusions
� Labeled cRNA from different levels of RNA degradation results in low Cy Dye incorporation and low yield of cRNA
� RNA integrity levels of starting material had serious impact on downstream gene expression microarray results
� The RIN is an effective tool that can be used to evaluate RNA integrity objectively
Page 51 2008
18S
28S
Flu
ore
sce
nce
Time (seconds)
0.0
2.5
5.0
7.5
10.0
12.5
16 21 26 31 36 41 46 51 56 61 66 71 76 81
18S
28S
Flu
ore
sce
nce
Time (seconds)
-0.5
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
5.0
5.5
21 26 31 36 41 46 51 56 61 66 71 76 81
rRNA contamination: 19.5 %
rRNA contamination: 1.7 %
Mou
se k
idne
y I
mR
NA
Mou
se k
idne
y II
mR
NA
Rat
bra
in m
RN
A
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ine
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RN
A
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se te
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se li
ver
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Ribosomal RNA contamination in mRNA samples
Page 52 2008
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Laser Microdissection – PALM MicroBeam System and RNA Pico kit
Data kindly provided by P.A.L.M. Microlaser Technologies
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Page 53 2008
Analysis of Small RNA (using RNA 6000 Assay)
Small RNA fraction: < 200 nts e.g. miRNA, siRNA, snRNA, tRNA, 5S RNA
Bioanalyzer allows discrimination of different profiles
Page 54 2008
Sm
all R
NA
Reg
ion
New Small RNA Assay versus existing RNA Assay
miR
NA
Reg
ion
Low
er M
arke
r
Small RNA Region
tRN
A
18s
28s
RIN: 8.1
Low
er M
arke
r
RNA 6000Nano kit
Small RNA Kit
RNA 6000Nano
Size range: 25-6000nt
Results: Integrity, Total RNA amount, gDNA contamination
NEW! Small RNASize range: 6-150nt
Results: miRNA amount, Ratio and amount of other Small RNA
5s
5.8s
Page 55 2008
ApplicationsThe new small RNA Assay as a tool for:
Verification, comparison and optimization in the small RNA region:
• High sensitivity to detect low abundant fragments• High resolution for ss oligos, miRNA, pre-, t-, 5S-RNA’s • compatible with Total RNA samples or purified small RNAs.• Semi-quantitative for single stranded RNA.• semi- Denaturing• Analysis up to 150nt
miR
NA
tRN
A
rRN
A
rRN
ALM
Intact Total RNA sample
(size separation and relative amount estimation)
Plus: Qualitative assessment of dsDNA, siRNA or other hairpin RNA up to 150bp
Page 56 2008
Small RNA Assay specifications
Analytical Range 6 -150 nt (to avoid overlap)
Sensitivity 50 pg/µl (diluted Ladder - 40 nt fragment; S/N > 3:1)
Quantitative range 50 pg/µl – 2000 pg/µl (purified miRNA in water after extraction ~<200nt)
Quantitation Reproducibility 25 % CV(defined on Ladder)
Max amount total RNA 100 ng/µl total RNA
Carryover Below detection limit
Page 57 2008
Protein Applications
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Page 58 2008
Bioanalyzer Protein Kit portfolio
Intro
ducti
on
P 80
Range 5 - 80 kDaSensitivity: CoomassieSamples: 10
Samples-Antibodies (reduced)-Small Proteins
P 230
Range 14 - 230 kDaSensitivity: CoomassieSamples: 10
Samples-Antibodies (all types)-Standard Proteins
Range: 10 - 250 kDaSensitivity: 1 pg/µl BSA on ChipSamples #: 10 per ChipChips #: 10 per KitLabeling Conc: 1 ng – 1 µg /µl
Coomassie Range (5 ng/µL BSA)
HSP 250
Silver stain Range (200 pg/µL BSA)
Agilent Protein 80 kit Prod Number 5067-1515Agilent Protein 230 kit Prod Number 5067-1517Agilent High Sensitivity Protein 250 kit Prod Number 5067-1575
Page 59 2008
Protein Kit Specifications
* Prior to measurement on Chip we recommend within the High Sensitivity Protein 250 labeling protocol to dilute the labeled sample by a factor of 200.
Product No. 5067-1515 Product No. 5067-1517 Product No. 5067-1575
New
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Page 60 2008
'������ (��������� ��������������Staining, Destaining and Detection (P-80 and P-230)
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Page 61 2008
Clone Selection based on Protein Expression
1* 2 3 4 5 6 7 8*
Fluo
resc
ence
Time (seconds)
0
10
20
30
40
50
60
20 25 30 35 40 45 50 55
colony 1 colony 2
53.0
21.5
29.032.5
14.4
6.0
kDa
protein of interest
Example measured withProtein 50 kit
Page 62 2008
Monitoring of Protein Purification Process
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Example measured withProtein 200 plus kit
Page 63 2008
Expression of a Recombinant Protein in E.coli- Optimization of Fermentation and Induction Conditions
6,00
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38
13,4
6
25,2
5
36,7
440
,63
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153,
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1,05
210,
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Fluo
resc
ence
Time (seconds)
-10
0
10
20
30
40
50
60
70
80
90
100
110
120
15 20 25 30 35 40 45
6,00
*
13,4
614
,84
22,7
5
32,2
1
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84,4
4
100,
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127,
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5,36
145,
76
210,
00*
Fluo
resc
ence
Time (seconds)
-10
0
10
20
30
40
50
60
70
80
90
100
110
120
15 20 25 30 35 40 45
recombinant protein
soluble protein fraction
solubilized inclusion bodies (50 mM Tris pH 7.5, 100 mM DTT, 8M urea)
Page 64 2008
Quality control of the Depletion of High Abundance Proteins in Human Serum
Depletion of High Abundance Proteins by Agilent MARS HPLC columns
*
Fractions checked by 2100
Page 65 2008
Quality Control of Antibodies
90 kDa 160 kDa
intact antibody
16% half antibody
Determine the half antibody content in IgG preparations
Antibody analysis under reducing and non-reducing conditions
Ab reduced
Ab non-reduced
light chain
heavy chain
intact antibody
light + heavy chain
Ab reduced
Ab non-reduced
Absolute Quantitation of IgG samples
Page 66 2008
Antib
ody
1 - S
tand
ard
Ladd
er
Antib
ody
1 –
1 m
onth
40°
C
Antib
ody
2 –
12 w
eeks
40°
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Antib
ody
2 - S
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ard
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Fluo
resc
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Time (seconds)
0
100
200
300
400
500
600
21 23 25 27 29 31 33 35
Antibody 1- Standard Antibody 1- 1 month 40°C
Fluo
resc
ence
Time (seconds)
0
100
200
300
400
500
600
21 23 25 27 29 31 33 35
Antibody 2- Standard Antibody 2- 12 weeks 40°C
Analysis of Antibody Stability – stress test
Page 67 2008
Combination of IEF with SDS-PAGEAgilent 3100 OFFGel Fractionator + 2100 bioanalyzer
Page 68 2008
Description of the new HSP-250 Assay(Direct labeling reaction, silver stain sensitivity)
• Reach and beat traditional „silver stain sensitivity“
• Offer solid quantitation for a large dynamic range
Target Applications:• Protein QA/QC
reliable quantitation of main compound besides minor impurities
• Protein detection at lowest concentrations in research
High Sensitivity Protein 250 Kit 5067-1575 content is:- 10 Chips (100 samples)- Labeling Kit (Dye and Reagents)- 2100 Separation Kit (Gel, Marker, Ladder, Buffer)- User Documentation (Quick Start Guide & Labeling Protocol)
Page 69 2008
Extended experimental workflow
Transfer to suitable buffer(precipitation, ultrafiltration, buffer exchange spin columns)
Labeling with dye(N-hydroxy-succinimidyl ester chemistry)
Sample preparation for 2100(SDS denaturation, dilution if desired)
Analysis on 2100
labeling kit
separation kit
5-90 min
40 min
5 min
35 min
Sample
Data
Page 70 2008
Principle of High Sensitivity 250 Protein Staining
pH 8.50°C
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NHS
NH2EtOH
10 min
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Step 1Labeling Reaction
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5 min
Step 2SDS-Denaturation
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Step 3Separation on Chip
Page 71 2008
Reproducibility of Labeling Reaction: Ladder
Rugged Labeling reaction:Reproducible reaction provides comparable signal intensities. Homogenous labeling without extra bandbroadening
No deviation in peak width is indicating a constant number of dye per protein molecule and proves a stable protocol
Page 72 2008
Sensitivity: Silver Staining vs. Bioanalyzer
Highest sensitivity:Labeled proteins can be measured down to pg/µL concentrations loaded on Chip
Direct comparison of samples run on SDS-PAGE with Silver staining and on 2100 Bioanalyzer.
Concentrations are given per lane (as total concentration of 7 different proteins)
Page 73 2008
Linear Dynamic Range Test: IgG
Linear dynamic range:Quantification of labeled IgG from 10 pg/µL to 100 ng/µLaverages ± SD of 7 measurements (7 chips, 1 chip lots, 4 instruments)
4 orders of linear dynamic range allows to quantify an 0.05% impurity besides the main peak in a single run
Page 74 2008
2100 Bioanalyzer Compliance
2100 expert software- One version for all assays- Declaration of system validation
2100 expert security pack- 21 CFR part 11compliance- Electronic records- Electronic signatures- Audit trails
2100 bioanalyzer - IQ and OQ/PV services- Declaration of conformity
Chips and reagents- Declaration of conformity
Page 75 2008
Agilent Web pages with 2100 content
www.opengenomics.com
www.agilent.com/chem/labonachip
Page 76 2008
2100 kits for Protein applications
Number Kit Max # of samples
5067-1517 Agilent Protein 230 Kit 250
5067-1518 Agilent Protein 230 Reagents
5067-1515 Agilent Protein 80 Kit 250
5067-1516 Agilent Protein 80 Reagents
Protein Kits – Coomassie stain sensitivity
Number Kit Max # of samples
5067-1575 High sensitivity Protein 250 Kit 100
5067-1576 High sensitivity Protein 250 Reagents
5067-1577 High sensitivity Protein 250 Labeling Reagents
5067-1578 High sensitivity Protein 250 Ladder
Protein Kits – Silver stain Sensitivity
Page 77 2008
2100 kits for Cell Assay and DNA applications
Number Kit Max # of samples
5067-1519 Agilent Cell Kit 150
5067-1520 Cell Checkout Kit
Cell Fluorescence Kits
Number Kit Max # of samples
5067-1504 Agilent DNA 1000 Kit 300
5067-1505 Agilent DNA 1000 Reagents
5067-1506 Agilent DNA 7500 Kit 300
5067-1507 Agilent DNA 7500 Reagents
5067-1508 Agilent DNA 12000 Kit 300
5067-1509 Agilent DNA 12000 Reagents
DNA Kits
Page 78 2008
2100 kits for RNA applications
Number Kit Max # of samples
5067-1511 Agilent RNA 6000 Nano Kit 300
5067-1512 Agilent RNA 6000 Nano Reagents
5067-1529 Agilent RNA 6000 Nano Ladder
5067-1513 Agilent RNA 6000 Pico Kit 275
5067-1514 Agilent RNA 6000 Nano Reagents
5067-1535 Agilent RNA 6000 Nano Ladder
5067-1548 Agilent Small RNA Kit 275
5067-1549 Agilent Small RNA Reagents
5067-1550 Agilent Small RNA Ladder
RNA Kits
Page 79 2008
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Flu
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Time (seconds)
0
100
200
300
400
500
25 30 35 40 45 50 55 60 65 70 75 80 85 90
3000 bp PCR 3000 bp PCR 1:4 dil.
Impurity level : > 50%3000 bp
Determination of PCR Product Impurity1* 2 3 4*
Flu
ores
cenc
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Time (seconds)
0
100
200
300
400
500
25 30 35 40 45 50 55 60 65 70 75 80 85 90
300 bp PCR 300 bp PCR 1:4 dil.
Impurity level : < 2%
300 bp
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