Simple Western validated antibodies
Simple Western Instruments
Simple Western Kit Builder
Simple Western Webinars
Protein Simple Technical Library
What is a Simple Western?
Simple Western is the first fully automated and complete solution for protein detection and characterization, representing a true reinvention of Western blotting. Simple Westerns separate proteins by size, and precisely manages immunoprobing, incubations, washes and even detection steps by removing the manual, error-prone steps in standard western blots. Simply load your sample, antibodies and reagents, and insert the sample plate and cartridge into your Simple Western instrument. Come back to fully analyzed results in as little as 3 hours, not 3 days. Compass software reports molecular weight, area, percent area and signal to noise for each protein detected. Sample data is displayed by lane in a virtual-blot like image similar to traditional results and an electropherogram view (example below).
Western Blot vs Simple Western: What is the difference?
Simple Western assays are gel-free, blot-free, and require approximately 1 hour of hands-on time. Simple Western automates all steps from protein loading and separation, immunoprobing, washing, detection and quantitative analysis of data. Simple Western platforms allow the separation and analysis of proteins with molecular sizes ranging from 2 - 440 kDa either via immunoblotting or total protein analysis on up to 96 samples at a time.
My antibody is validated for Western Blot. Does that mean it's applicable for Simple Western?
Many of the antibodies used for traditional Western blot can also be used for Simple Western platforms. However, antibodies will still require optimization and validation to be used for the Simple Western application.
How can I find a Simple Western validated antibody?
The first step in migrating a traditional Western assay to a Simple Western instrument is to select primary antibodies already validated for the application when possible. We recommend starting your search among the over 1,300 antibodies validated for Simple Western by Novus. View our complete portfolio of Simple Western validated antibodies. If your antibody hasn’t been validated for Simple Western, you can submit your successful results to our Innovator's Reward Program.
What should I do if my Simple Western validated antibody did not work?
If you are having trouble with your target of interest using a validated antibody, you should email technical support at firstname.lastname@example.org. Our technical team will be happy to help you investigate the issue and find a potential resolution.
How much sample do I need for Simple Western?
Simple Western analysis involves smaller wells in a capillary electrophoresis setting and it uses as little as 3 µL in Wes and 5 µL of the sample per capillary in all other instruments. Sample concentration is typically between 0.1 and 2.0 mg/mL and of that sample, 3-5 µL is pipetted into the well.
What antibody concentration should be used for Simple Western?
As with all experiments, every antibody should be titrated to suit your specific sample. Validated antibodies will likely give you a starting concentration. For antibody optimization/validation, it is recommended to start with 10 to 50-fold higher antibody concentration than what is used for traditional Western blot. Example: If your western blot requires 1 µg/mL, you would use at least 10 µg/mL or ratios of 1:1000 becomes 1:100.
What tissue homogenization buffer can be used for Simple Western?
Protein Simple has done extensive compatibility testing with multiple buffers and reagents. You can view their buffer compatibility table to assess your reagents.
How is protein normalization performed in Simple Western?
Housekeeping genes can be an unreliable choice for normalization since their expression can be influenced by various factors. Normalizing target protein abundance to the overall amount of protein present in a sample is a more accurate means for eliminating technical errors and determining fold-change in protein expression. Protein normalization is antibody-independent and minimizes the impact of varying expression of a loading control. Using a proprietary in-capillary protein normalization (PN) reagent, instruments like Jess™ can normalize your protein with ease. The fluorescent reagent binds to proteins immobilized in the same capillary as your immunoassay via primary and secondary amine interactions. ProteinSimple recommends performing a titration curve ranging from a lysate concentration 0.05 to 2 mg/mL of both the PN Reagent and target protein to determine optimal concentrations within the same linear range. Learn more about protein normalization.
How many targets can I assess through Simple Western?
Multiplexing takes your options to the max, helping you identify and differentiate relative protein expression between samples in one shot. Like traditional Western blots, Simple Western can multiplex any number of different molecular weight (MW) targets as long as differentiated host species are used for the antibodies. What sets Jess apart from the rest is she can multiplex across chemiluminescence, NIR and IR channels simultaneously allowing for the detection of up to 3 identical MW targets through these 3 channels. You can now leverage the great sensitivity of chemiluminescence for your low abundant proteins with the multiplexing capabilities of NIR/IR for your other targets of interest.
What instrument is best used for my sample population?
Using Simple Western, you separate and analyze proteins by size from 2-440 kDa either by immunoassay or total protein analysis. To analyze proteins by size, choices include Jess, Wes™, Sally Sue™ and Peggy Sue™.
When performing post-translational modification characterization, the charge-based Peggy Sue or NanoPro 1000™ instruments are best. These systems automate all steps including sample loading, charge-based protein separation, immunoprobing, detection and data analysis. Only requiring as few as 25 cells per assay, Peggy Sue or NanoPro 1000 can quantify low abundance proteins in limited cell populations in up to 96 samples at once.
Simple Western protocols: Size or charged-based assays explained
ProteinSimple’s interactive Kit Builder makes picking the right modules for your assay fast, easy and unique for any instrument or sample type you need.
What should I expect from a size-base assay Simple Western?
To analyze proteins by size, samples and reagents are loaded into an assay plate and placed into instruments Jess, Wes, Sally Sue or Peggy Sue. Sample is loaded into the capillary automatically, electrophoresed and separated by size as they migrate through a stacking and separation matrix. The separated proteins are then immobilized to the capillary wall via a proprietary, photoactivated capture chemistry. Target proteins are identified using a primary antibody and immunoprobed using an HRP-conjugated secondary antibody and chemiluminescent substrate. Sample data is displayed by lane in a virtual-blot like image similar to traditional results and an electropherogram view. The resulting chemiluminescent signal is detected and quantitated. Quantitative results such as molecular weight, signal intensity (area), percent area, and signal-to-noise for each immunodetected protein are presented in the results table automatically from Compass software.
What should I expect from a charged-base assay Simple Western?
To assess protein separation by charge, samples and reagents are loaded into an assay plate and placed into Peggy Sue or NanoPro 1000. Your sample loads into the capillary automatically, is separated by charge and proteins resolve according to their isoelectric point (pI). The pI is the pH of a solution at which the net charge of a protein becomes zero. At solution pH that is above the pI, the surface of the protein is predominantly negatively charged, and therefore like-charged molecules will exhibit repulsive forces. The separated proteins are then immobilized in the capillary via a proprietary, photoactivated capture chemistry. Target proteins are identified using a primary antibody and immunoprobed using an HRP-conjugated secondary antibody and chemiluminescent substrate. The resulting chemiluminescent signal is detected and quantitated. Your output will be electropherograms, which make it easier to see minute changes. Quantitative results such as pI value, signal intensity (area), percent area, and signal-to-noise for each immunodetected protein are presented in the results table automatically from Compass software.
Why do I observe a shift in the molecular weight of proteins?
Like traditional Western blot assays, the Simple Western application does not provide absolute molecular weight (MW) but an observed MW. When compared to the predicted MW of a protein, the observed MW in these applications are influenced by many factors such as the pH of the running buffer as well as the nature of the stacking and separation matrix or gel, in addition to post-translational modifications. See ProteinSimple's Technical Note on how SDS denaturization impacts molecular weight of proteins.
What software do I need for Simple Western instruments?
You will need to download the free Compass for Simple Western software. Compass will analyze Simple Western Size or Charge-based assay data and process the results for you. Data can be displayed in a lane view, graph view, or as a digital capillary image and can be easily shared.