1. The Simple Western is Here Name Date

2. 2

3. Western Blots are Tedious 3

4. Western Blots are Inconsistent 4 Day 1 Day 2

5. Meet Simon Gel-free, blot-free, hands-free Simple Western Assays 5

6. Simon Gives You More Walk away assay convenience Fully analyzed data Ease of Use Automated start to finish Protocol standardization Accurate quantitation Greater reproducibility Automation Higher Data Quality 6

7. Simple Westerns are Easy 7 Prepare samples Start Simon Results automatically

8. Get More Information 8 MCF7 Ladder Traditional Western BlotSimple Western

9. More Quantitative 9

10. More Reproducible 10 CV=5.2%CV=11.5%CV=12.0%CV=7.3%CV=9.7% 14000 12000 10000 8000 6000 4000 2000 0 egarevAyadsruhTyadsendeWyadseuTyadnoM NickNancyTimJessica Experiments(n=11replicates) TargetProteinReproducibility Signal

11. Global Standardization 11

12. How Simple Westerns Work Immobilize Separate Immunoprobe Quantitate Signal

13. Limits of Detection 13 250 150 100 75 50 37 25 20 15 10 Western Blot Simple Western 2.0 mg/mL1.00.50.250.1250.0640.0320.0160.008 KDa Ladder 2.0 mg/mL1.00.50.250.1250.0640.0320.0160.008 Ladder

14. Wide Dynamic Range of Detection 14 Ladder3.0 mg/mL1.00.330.110.030.010.0030.0010.00030.0001 KDa

15. Multiplexing Capabilities 15 Courtesy of Senior Scientist pharmaceutical evaluator AKT2 Thioredoxin Ladder Rep 1 Rep 2 Rep 3 Rep 4

16. Comprehensive Pathway Validation 100 Targets Detected Insulin/ AKT NF  B DNA Damage Cell CycleApoptosis JAK/ STAT EGFR 16

17. Monitoring Biological Response Phospho-STAT5Total STAT5 - + MW - + MW 17 Courtesy of Senior Scientist pharmaceutical evaluator

18. Simple Westerns Offer More FeatureSimple WesternWestern Blot Quantitation Reproducibility Automation & Time Savings Sensitivity Throughput 18

19. Complete Solution InstrumentKitsSoftware 19

20. 20 Powering Protein Research PurityImagingSignalingAggregationCharacterization

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22. Supplemental Slides 22

23. How the Simple Western Works 23

24. Simple Western Assay Specs DescriptionSpecification Samples per run12 Sample required3 µg Total run time3-5 hrs Sizing range15 - 150 kDa* Sizing accuracy± 20% across sizing range Sizing CV10% Resolution± 10% difference in molecular weight at 50% baseline (similar to resolution on a 4-20% gradient or 10% non-gradient SDS- PAGE gel) Dynamic range3 logs Relative Signal Quantitation CV < 20% across sizing range SensitivityLow ng *Assay specifications apply only to the stated sizing range. However, proteins up to 200 kDa can be detected.