The Simple Western is Here
Name
Date
2
Western Blots are Tedious
3
Western Blots are Inconsistent
Day 1
Day 2
4
Meet Simon
Gel-free, blot-free, hands-free
Simple Western Assays
5
Simon Gives You More
Ease of Use
Walk away assay convenience
Fully analyzed data
Automation
Automated start to finish
Protocol standardization
Higher Data
Quality
Accurate quantitation
Greater reproducibility
6
Simple Westerns are Easy
Prepare
samples
Start
Simon
Results
automatically
7
Get More Information
Traditional Western Blot
Simple Western
8
More Quantitative
9
More Reproducible
Target Protein Reproducibility
14000
12000
Signal
10000
8000
6000
CV=5.2%
CV=11.5%
CV=12.0%
CV=7.3%
CV=9.7%
Monday
Nick
Tuesday
Nancy
Wednesday
Tim
Thursday
Jessica
Average
4000
2000
0
Experiments (n=11 replicates)
10
Global Standardization
11
How Simple Westerns Work
Separate
Separate
High MW
Low MW
Target Protein
Primary Ab
HRP-labeled
Secondary Ab
Immobilize
Immobilize
Immunoprobe
Immunoprobe
Quantitate
Quantitate
Signal
Signal
Limits of Detection
250
KDa
150
100
75
50
37
25
20
15
10
Simple Western
Western Blot
13
Wide Dynamic Range of Detection
1000000
Dynamic range for GSK3 Alpha
KDa
10000
Peak Area
100000
1000
100
R² = 0.9995
10
0.0001
0.001
0.01
0.1
1
10
K562 Lysate Concentration [mg/ml]
14
Multiplexing Capabilities
Reproducibility of Data
14
AKT2
12
10
%CV
8
6
4
Thioredoxin
2
0
AKT
Thioredoxin
AKT/Thioredoxin
Courtesy of Senior Scientist pharmaceutical evaluator
15
Comprehensive Pathway Validation
Insulin/
AKT
NFkB
EGFR
JAK/
STAT
100
Targets
Detected
Apoptosis
DNA
Damage
Cell Cycle
16
Monitoring Biological Response
MW -
+
MW
- +
Courtesy of Senior Scientist
pharmaceutical evaluator
Total STAT5
Phospho-STAT5
17
Simple Westerns Offer More
Feature
Simple Western
Western Blot
Quantitation
Reproducibility
Automation &
Time Savings
Sensitivity
Throughput
18
Complete Solution
Instrument
Kits
Software
19
Powering Protein Research
Signaling
Purity Characterization Aggregation
Imaging
20
21
Supplemental Slides
22
How the Simple Western Works
Load Matrix
Stacking Matrix
Separation Matrix
Load Sample
High MW
Low MW
Separate
Immobilize
Immunoprobe
Target Protein
Primary Ab
HRP-labeled
Secondary Ab
Quantitate
Signal
23
Simple Western Assay Specs
Description
Specification
Samples per run
12
Sample required
3 µg
Total run time
3-5 hrs
Sizing range
15 - 150 kDa*
Sizing accuracy
± 20% across sizing range
Sizing CV
10%
Resolution
± 10% difference in molecular weight at 50% baseline (similar
to resolution on a 4-20% gradient or 10% non-gradient SDSPAGE gel)
Dynamic range
3 logs
Relative Signal Quantitation
CV
< 20% across sizing range
Sensitivity
Low ng
*Assay specifications apply only to the stated sizing range. However, proteins up to 200 kDa can be detected.