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Why Choose Simple Western for Western Blot Protein Quantification

Choosing a housekeeping control for accurate protein quantification can be challenging. Many housekeeping controls fluctuate in response to cell stimuli or cell state, producing misleading normalized results that undermine research quality. Instead, normalization by total protein is the most reliable way for accurate protein quantification since it normalizes out experimental variation in protein load without being subject to biological variations. Now, total protein normalization (TPN) is built-in with Simple Western™, the next generation in Western blotting protein analysis from ProteinSimple™, a Bio-Techne brand.

  • The only fully automated protein normalization Western blotting platform
  • Quantitative and reproducible results in as little as 3 hours
  • Tiny sample volumes, as little as 3 µL
  • Use conventional Western blot antibodies
  • Built-in TPN in chemiluminescence or fluorescence channels
  • Multiplex detection with industry-leading sensitivity using chemiluminescence and/or Stellar NIR/IR modules

The figure on the right shows multiplex detection of Actin and HSP60 along with total protein in the same lane for more accurate immunoassay quantitation on the Simple Western instrument Jess.

Multiplex detection of Actin (red) and HSP60 (green) with total protein detection (blue) on Jess.

Transition from Housekeeping Proteins to an Automated Total Protein Normalization Western Blotting Platform

Simple Western makes normalizing protein expression by total protein easy, so you can leave behind normalizing by unreliable housekeeping proteins for good. Shown here are two methods for TPN by channel-based TPN or sequential TPN on the same 3 µL of sample. Both strategies are automated, generating reproducible and fully quantitative results in just a few hours.

IR: Infrared, NIR: Near-infrared, HRP: Chemi, FL-TPN: Fluorescence TPN. Not shown: Stellar TPN

Channel-Based Total Protein Normalization

While chemiluminescence detection comes standard with all Simple Western systems, Jess provides additional protein detection flexibility in IR/NIR fluorescence channels, pushing the boundaries of multiplexing by enabling a variety of strategies for the detection of multiple proteins in each sample. Jess even has a separate fluorescence channel dedicated to TPN that is performed simultaneously in the same capillary. Now, Jess has industry-leading fluorescence detection sensitivity for Western blot analysis with the Stellar NIR/IR Modules that can be paired with Stellar™ TPN Assays.


IR: Infrared, NIR: Near-infrared, HRP: Chemi, HRP-TPN: Chemi TPN

Sequential Total Protein Normalization

Both Jess and Abby replace the laborious strip and re-probe of the traditional Western blot with RePlex™ from ProteinSimple, a Bio-Techne brand. RePlex efficiently removes antibodies from the capillary for a second round of immunoassay with fresh antibodies. The second cycle of RePlex may also be dedicated to chemiluminescence-based total protein detection, so you can normalize your protein expression data with confidence.

Total protein normalization Western blot assay capabilities on Simple Western

Tubulin or Not Tubulin: The Advantages of TPN

In this on-demand webinar hosted by labroots, Dr. Moritz discusses:

  • The advantages total protein staining has over classical housekeeping proteins when used as a loading control in Western blots
  • How technical and biological variation is reduced when using total protein staining
  • Total protein normalization Western blot assay capabilities on Simple Western
White paper detailing how Protein Simple helps researchers meet publication standards in protein normalization

Meeting New Scientific Standards with Publication-Ready Results

Producing publication-quality data with Western blot is challenging and prone to misrepresentation. Simple Western changes that by generating fully quantitative and reproducible results that are not prone to misrepresentation.

This White Paper examines how Simple Western overcomes the common pitfalls associated with traditional Western blots to produce publication-ready data that meets the highest standards of data integrity and rigor, including Total Protein Normalization.