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Why do Protein Normalization?

Planning on publishing your research findings? Then you know scientific journals and peer reviewers need to see some type of total protein characterization that validates your data. They're also asking scientists to move away from housekeeping proteins (HKP) because their expressions are inconsistent and unreliable. That's where protein normalization (PN) assays on Jess come in. They give you a more consistent, robust way to determine protein content.

 

Figure 1 (Right): Comparative total protein data and the expression of the housekeeping protein β-actin in six human whole tissue lysates loaded on an equal protein basis (0.3 mg/mL).

B-actin graph
Protein Normalization vs Housekeeping Proteins

Meet Jess

Gel-free, blot-free, hands-free Simple Westerns™ let you separate and analyze proteins by size from 2–440 kDa either by immunoassay or total protein analysis. You'll get quantitative results, reproducibility that's spot on, and use less sample in the process.

Experimental prep only takes 30 minutes—just load your samples and reagents into our microplate. Jess handles the rest: antibody additions, incubations, washes, immunodetection and analysis. Fully analyzed results are ready in 3 hours.

Jess instrument by ProteinSimple a Bio-Techne brand

Traditional Western Blot vs. Simple Western PN

 

Here's what you don't have to deal with:

There's also some added bonuses:

Hands-on staining Keep all your detection channels
 Wash steps Only one reagent
Gels or membranes Get going in seconds
Methanol Completely automated assays

 

How Simple Western PN Works

With Jess, you can run PN assays with the immunoassays you'd already be doing. It's just one extra reagent to mix, and one extra row to pipette on the plate. Jess needs an extra 30 minutes to run the PN assay for you, but once she's done you'll have all the data you need.

PN assay label binds reliably to any amine group

  • Proprietary molecule developed with Tocris Bioscience
  • No antibody interference
  • No cross-talk
Biomolecule
Capillary diagram

PN, IR, NIR and chemiluminescent detection at the same time

Total protein detection happens in the same capillary on the same sample, but runs on a separate channel from NIR so you get to keep your existing IR, NIR and chemiluminescence channels. Jess' same-time detection also gives you more consistent, reliable results than traditional assays without having to stain and wash a single blot!

One-Stop Data Processing

Compass software gives you all the processing tools you need for protein analysis and normalization.

Automatic Data Analysis

Once the assays are done, no need to export the data for analysis. Jess' Compass software analyzes the data for you. It also transforms your raw data into normalized data automatically.

Compass application
User interface
Gel lane view

Only with Compass: Overlay PN with Your Targets

Compass lets you look at all your data with a single click: traditional lane view, quantitated total protein values or relative differences to your reference point.

 

Figure (Left): Lane view of protein normalization on Jess in Compass for Simple Western Software. Shown are three options for visualization: the traditional total protein "membrane stain" (left); dot overlay of the raw total protein area measured in each "lane" (middle); dot overlay of the normalized percent total protein area measured relative to the chosen reference "lane" or capillary (right), in this case the 1 mg/mL sample.

Export and Done

Compass export options let you pick multiple formats to process your data using external programs like Microsoft© Excel©, Spotfire, JMP and others.

 

Figure (Right) Comparative data showing 14-3-3 gamma protein expression (blue) and the normalized counterpart (orange) in various concentrations of Jurkat cell lysate.

Export Graph

Simple Westerns on Jess

Download our Tech Note to learn more about protein normalization on Jess. Did you know she also gives you four different ways to analyze proteins—simultaneously?

  • Fluorescence detection with multiplexing gives you all the info you need in one shot—no stripping and reprobing.
  • Chemiluminescence detection for the picogram-level sensitivity you need on those low abundance proteins and precious samples.
  • Protein normalization lets you see if your samples contain a consistent protein load.
  • Blot imaging whenever you want to run a traditional chemiluminescent Westerns.
Characterize post transduction protein expression using automated Simple Western machine Jess