Simple Western™ is a CE platform that pairs protein size or charge separation with a quantitative immunoassay readout and represents the next-generation gold standard for quantitative, reproducible, and highly specific analytical-grade protein expression potency assays. Plus, Simple Western is a multi-attribute method used in cell and gene therapy process development for quantitative CQA monitoring of purity, titer, empty/full capsid content, and capsid protein ratio.
- Quantitation. Quantitative protein measurements with a 4-log dynamic range.
- Sensitivity. Conserve your precious AAV sample with CQA analysis, including purity assays that are more sensitive than SYPRO Ruby.
- Reproducibility. Eliminate the variation of traditional Western blots -CVs below 15-20%.
- High Throughput. Up to 96 data points in a single overnight run, all in a fully automated fashion.
- Method Transferability. Easily transfer between sites or from sponsors to CROs. 21 CFR Part 11-compliant and GMP-ready.
The New Gold Standard for Transgene Expression Assays
Get the Flexibility of Western Blots with the Analytical-Grade Quantitation of ELISA
By pairing the flexibility and specificity of Western blots with the analytical-grade quantitation of ELISA, Simple Western is ideal for making specific measurements in complex sample types like tissue homogenates and analyzing intracellular protein expression without matrix effects that can confound ELISA read-outs. As a platform technology that needs only a single validated antibody and offers rapid 3-hour runtimes, Simple Western lowers the custom assay development hurdles of ELISA and accelerates drug development from pre-clinical phases and scaling to GMP. In this Application Note, we demonstrate a Simple Western relative potency assay with parallel line analysis (PLA) for AAV-mediated gene delivery in cultured human cells.
One Platform, Many Assays to Support Your Process and Product
3 Hours to Results, Just 3 µL of Sample
Single-Cell Westerns on Milo for Heterogeneity Analysis
Single-Cell Westerns on Milo measure protein expression in thousands of single cells per run. Their high sensitivity combined with the ability to use conventional Western blotting antibodies makes them particularly well-suited for quantifying gene therapy efficacy and tracking stem cell differentiation.
Milo's Single-Cell Westerns are used in clinical trials aimed at advancing gene therapies for Sickle Cell Disease and other hemoglobinopathies. Milo resolves hemoglobin subunits in single red blood cells, enabling researchers to track the efficacy of sickle cell gene therapies.
Milo can identify and quantify neural subtypes in a heterogeneous neural sample, and monitor the differentiation of iPSCs into neurons, astrocytes, and oligodendrocytes using our research-grade or GMP differentiation reagents.
|Title||Key Data Sets||Take-Home Message|
|The New Gold Standard for Protein Expression Potency Measurements||(1) Relative protein expression potency measurements between AAV serotypes using parallel line analysis (PLA)
(2) Absolute protein expression measurements using a recombinant standard curve
(3) Quantification of AAV VP1/VP2/VP3 capsid proteins in transduced cells as a surrogate for AAV infectivity
|Simple Western provides analytical-grade protein expression quantification for relative potency assays in a multi-attribute platform approach. AAVs can be detected in transduced cells as a surrogate for infectivity.|
|Do Your AAVs Contain DNA - Rapid and Sensitive AAV Empty/Full Ratio Assay with Simple Western||(1) Empty/full content ratio of AAVs and assay linearity, reproducibility, and specificity
(2) Platform method approach for multiple AAV serotypes
(3) Empty/full measurements of intact AAVs using Simple Western Charge as an orthogonal method
|Simple Western provides automated empty/full AAV capsid content ratio measurements with high sensitivity, reproducibility, and scalability. Simple Western is a multi-attribute platform method to characterize multiple AAV serotypes in complex sample types.|
|AAV2 VP Protein Standards and Their Use In Quantifying Capsid Protein Ratio by Western Blot and Simple Western||(1) Establishment of an AAV2 VP1, VP2, and VP3 Protein Standard (PROGEN)
(2) Analysis of VP1, VP2, and VP3 for capsid protein ratio and titer measurements
(3) Demonstration of Simple Western reproducibility and linearity and comparison to traditional Western blot
|PROGEN’s recombinant AAV2 VP protein standards are useful for accurate and sensitive analysis of VP content and VP ratios of AAVs. Simple Western removes the variability of traditional Western blot, plus advantages like automation, short time to results, tiny sample volume requirements, and high sensitivity.|
|Concentrating on AAV Impurities with Ultrasensitive Total Protein Detection on Simple Western||(1) 5X biotin labeling reagent increases DnaK detection sensitivity
(2) RNase A spiked into purified AAV sample and analyzed with 1X and 5X total protein labeling reagent on Jess
(3) Multi-attribute identity and purity measurements of AAVs using RePlex
(4) Comparison of 5X biotin labeling reagent to SYPRO Ruby
|The 5X biotin labeling reagent provides ultrasensitive and reproducible total protein measurements in the upper picogram range, beating SDS-PAGE staining techniques like SYPRO Ruby and silver stain, which require lower nanogram levels for reliable detection.|
|Simple Western Analysis of Adeno-Associated Virus (AAV) Proteins||(1) Identification of AAV2 purification using VP-specific antibodies
(2) Range of detection of the anti-VP1/VP2/VP3 antibody
(3) Immunoassay and total protein analysis of AAV during purification from HEK293 whole-cell lysate
|Simple Western is a powerful analytical tool throughout the AAV product development pipeline, providing purity and identity measurements to monitor and characterize AAV capsids during product purification from HEK293 cells.|
|The Simple Western Assay for Residual Benzonase Quantification||(1) Recombinant standard curve for Benzonase quantification
(2) Calibration of the Benzonase assay with QC samples (% recovery)
(3) Quantification of residual Benzonase in HEK293T whole-cell lysates (<1 ng/mL)
and % recovery
|Simple Western overcomes the challenges of traditional ELISA without compromising sensitivity and reduces hands-on time to just 30 minutes for sample preparation and plate loading, making it possible to integrate at-line or near-line and achieve fast, actionable results.|
|Immune Cell Therapy|
|Title||Key Datasets||Take-Home Message|
|Lentiviral Vector Analysis Made Simple||(1) p24 titer measurements and cross-reactivity with other lentiviral proteins
(2) Lentiviral empty/full capsid content and stability assays
(3) 5X total protein labeling reagent versus anti-HEK293T HCP antibody
(4) Lentiviral transduction of iPSC-derived macrophages (customer data)
|Simple Western is an automated multi-attribute platform for scalable at-line lentiviral analytics, including p24 titer, identity, purity, stability, capsid content ratio, and transduction measurements.|
|Title||Key Datasets||Take-Home Message|
|Simple Western Unravels the Signaling Web in Stem Cells for Regenerative Medicine||(1) Quantification of stemness biomarkers (Nanog, OCT3/4, SOX2) in naïve iPSCs and iPSC-derived endoderm, ectoderm, and mesoderm cells
(2) Multiplex analysis of total and phospho-protein isoforms (Akt and Smad1/5) plus total protein normalization in naïve iPSCs and iPSC-derived mesodermal cells in a single run using Stellar on Jess, RePlex on Abby, and Peggy Sue
|Using as little as 3 μL of starting material, Simple Western assays generate protein expression profiles of stem cells with flexible multiplex strategies and unprecedented sensitivity and reproducibility, revealing new insight into intracellular signaling cascades in response to iPSC differentiation.|
Customer Success Stories
Early Development Scientist, Oxford Biomedica
Senior Postdoctoral Researcher, University of Oxford
Centre for Tissue Engineering & Regenerative Medicine, UKM