> Simple Western Assays > Simple Western Size Assays > Simple Western Size-Based Total Protein Assays
Interested in looking at all the proteins in your lysate? Simple Western systems can do the work for you. Use your total protein data to identify impurities or contaminants in your sample or normalize your immunoassay data with total protein measurements for more reliable results.
Total protein normalization is a technique where target protein abundance is normalized to the overall amount of protein present in a sample rather than a housekeeping protein to minimize the impact of varying expression of a loading control. It is increasingly required for western blotting data by academic journals and grant agencies due to the improved reliability of normalized expression values. Download our white paper to learn more about total protein normalization.
There are two detection options available for total protein assays on Simple Western: chemiluminescence or fluorescence.
Chemiluminescent Total Protein Assays
Chemiluminescent total protein assays can be used to detect all the proteins in your lysate, similar to a Coomassie or SYRPO Ruby stained gel. Sample preparation, separation, and immobilization for the chemiluminescent Total Protein Assay are exactly the same as a Simple Western immunoassay. But, instead of specifically detecting your protein of interest with a primary antibody, the Total Protein Assay attaches biotin to all the proteins in your sample. Incubation with streptavidin-HRP followed by Luminol/Peroxide generates a chemiluminescent signal for total captured protein. The Total Protein Detection Module includes the reagents required to do a chemiluminescent total protein assay (Separation Module sold separately).
Total Protein detection. Decreasing concentrations of DNAK in Hela lysate and negative controls (15, 7.5 and 3.75 µg/mL).
The sensitivity of Total Protein Assays can be adjusted based on the needs of your sample by adjusting the concentration of biotin labelling reagent. Using 5-times more concentrated biotin labelling reagent, total protein detection using Simple Western can surpass the sensitivity of traditional protein stains including SYPRO Ruby. While SYPRO Ruby requires at least 1 ng of protein for reliable detection, Simple Western Total Protein Assays using 5x biotin labelling reagent can reliably detect as little as 150 pg. Download the total protein assay for AAV analysis app note to learn more about the use of ultra-high sensitivity total protein assays for AAV analysis used in the development of cell & gene therapies.
Side-by-side comparison of AAV total protein assay using SYPRO Ruby (right) and 5x labeling reagent with Simple Western (left) shows that Simple Western surpasses the sensitivity of traditional protein stains.
Chemiluminescent total protein assays can also be used to normalize immunoassay data using RePlex on Abby or Jess instruments. For samples containing 0.005 - 0.2 mg/mL of protein, chemiluminescence total protein assays can get you accurate normalized immunoassay data without having to rely on variable housekeeping proteins. For samples containing 0.2 – 1.2 mg/mL of protein, a fluorescent total protein assay can be run on Jess using the Protein Normalization Assay Module (see next section on Fluorescent Total Protein Assays).
Total Protein Normalization Using RePlex
Fluorescent Total Protein Assays
The Protein Normalization Assay Module on Jess enables you to run total protein assays in the same capillary on the same sample, without sacrificing your NIR, IR or Chemiluminescent detection channels. After separation & immobilization of your sample proteins, the Protein Normalization Assay Module for Simple Western uses a proprietary molecule to fluorescently label proteins in your sample for detection. The Protein Normalization Assay Module is appropriate for samples containing 0.2 – 1.2 mg/mL of protein and comes with both a separation module and the reagents required to do a total protein assay.
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