Simple Western systems
Gel-free, blot-free, hands-free
Bringing Traditional Western Blotting Into the 21st Century
What Is the Difference Between Simple Western Automated Western Blot Systems and Traditional Western Blot Workflows?
Simple Western platforms redefine your western blotting workflow by fully automating all assay steps including separation, immunodetection and data analysis. Simple Western platforms are based on capillary electrophoresis and Simple Western assays are performed in capillaries where SDS/heat-denatured proteins are separated by apparent molecular weight, immobilized covalently via a UV sensitive coating in the capillary, detected directly in the capillary by primary and secondary antibodies and analyzed by the integrated Compass Software.
By integrating and automating these previously manual workflow steps, Simple Western platforms overcome many of the limitations associated with traditional Western blotting workflows, significantly reducing hands-on time, and providing excellent reproducibility and assay transferability. Simple Western assays are quantitative, enabling generation of full dose response curves, and also include built-in total protein normalization so you can easily and accurately normalize your immunoassay data.
Simple Western is easy to use and also offers higher throughput and faster time to results. With only 30 min set-up times and walk away automation, you can get fully analyzed western blotting data on 25 samples in less than 3 hours on Jess, Abby, and Wes. If you want even higher throughput, Sally Sue or Peggy Sue can analyze up to 96 samples in a single run!
With all these advantages, Simple Western is rapidly becoming accepted as the preferred replacement for traditional Western blot protocols.
|Simple Western System||Jess||Abby||Wes||Sally Sue||Peggy Sue||Traditional Westerns|
|Maximum samples per run||25||25||25||96||96||25|
|Run time for mass samples (size assays)||<3 hours||<3 hours||<3 hours||<14 - 19 hours||<11 - 19 hours||2 days|
|Hands-on time||30 minutes||30 minutes||30 minutes||1 hour||1 hour||3.25 hours|
Simple Western – Your Partner for Publication Perfect Data!
Increasingly, the scientific community is setting new and more rigorous standards for data reproducibility, transparency and rigor in grant submission guidelines and journal publications. With over a thousand publications and counting, Simple Western systems overcome the challenges associated with traditional western blotting workflows to produce publication-ready data that meet the highest standards of data integrity, reproducibility and rigor.
Key Benefits of Using Simple Western for Publication-ready Data:
- Automated Simple Western platforms provide reproducible data, making it easy for other labs to reproduce & verify published results.
- Generate complete electronic data files which include publication-quality images and quantitative results as well as all assay conditions, making it easy to upload to data repositories to meet data sharing requirements.
- Perform total protein normalization in the same capillary as your immunoassay for more accurate results so you don’t have to rely on variable housekeeping proteins.
- Perform reliable sequential immunoassays in the same capillary with RePlex™ rather than struggling with variable stripping & re-probing protocols.
- Optimize your assay within hours making use of the vast selection of Simple Western certified antibodies.
- Reliably detect low abundance proteins using highly sensitive chemiluminescence or industry leading fluorescence detection sensitivity with Stellar NIR and IR modules.
- Get multiplexed western blotting data in the same sample using Jess’ fluorescence capabilities with NIR and IR, Stellar NIR and IR, and chemiluminescence channels.
MEET YOUR WESTERN PROTEIN ANALYSIS PROBLEM SOLVERS!
Two ways to get separation, identification and quantitation
You can separate your proteins two ways with Simple Western™ assays—by size or by charge. We've got systems that'll do one or the other, or both! No matter what option you choose, you'll get the separation you need, identification of your target protein, and truly quantitative data that lets you make the most accurate experimental decisions. Learn more.
Simple Western size-based western blotting assays
- A Simple Western by size is an automated Western—no gels, no transfer devices, no blots, no film and no manual analysis. Just load your samples in Jess, Abby, Wes, Peggy Sue, or Sally Sue and press start—it's a complete, walk-away solution for protein separation and detection.
- Jess, Abby, Wes, Peggy Sue, and Sally Sue automate all of the steps of traditional western blots by automatically including sample loading, size-based protein separation, immunoprobing or total protein labeling, washing, detection and data analysis.
- Western blotting automation eliminates variability inherent to manual processes which limits reproducibility, while adding quantitation, and improving on time-to-result and overall data reliability.
- The transition from traditional western blotting methods to automated western blots on Simple Western is simple—the same antibodies used for western blots can be used with our automated Simple Western assays.
- Up to 96 samples can be processed at once, and Simple Western assays take just 3-19 hours to complete with sizing and quantitation results.
- Separate and detect proteins as small as 2 kDa or as large as 440 kDa in only 3 hours.
How do Simple Western size-based assays work?
There are two ways to run Simple Western size-based assays—immunoassay or total protein. Just load your samples, start the instrument, and off to the races you go!
Simple Western immunoassays take place in a capillary. Samples and reagents are loaded into an assay plate and placed in Jess, Abby, Wes, Peggy Sue, or Sally Sue. As little as 40 nL of sample is loaded into the capillary automatically and proteins are separated by size as they migrate through stacking and separation matrices. 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. The resulting chemiluminescent signal is detected and quantified.
Interested in looking at all the proteins in your lysate? Just leave out the antibody and let Jess, Abby, Wes, Peggy Sue or Sally Sue do the work for you to add western blotting results of total protein in your sample. Target proteins separated by size are labeled with a biotin reagent and are detected by chemiluminescence using Streptavidin-HRP. At the end of the run, you've got relative quantitation for your protein of interest and total protein measurements of the sample.
What does Simple Western size data look like? Simple Western size-based assay data is processed automatically for you in Compass software. Sample data is displayed by lane in a virtual-blot like image similar to traditional western blot results with one big exception—not only do you get more information, you get it as soon as the assay is complete. Quantitative results such as molecular weight, signal intensity (area), % area, and signal-to-noise for each immunodetected protein are presented in the results table automatically.
Just need total protein? We've got that covered too!
If you're more familiar with capillary electrophoresis, you can see your results in a more traditional electropherogram view too.
Looking at big proteins? Try this on for size. Simple Western now has a wider molecular weight range capability, so you can detect proteins as large as 440 kDa.
Reproducibility and quantitation
Simple Western assays are fully automated by Jess, Abby, Wes, Peggy Sue, or Sally Sue and easily standardized, making your results much more reproducible. And there\’s no blotting step, so inconsistencies caused by protein transfer are eliminated, which really improves your quantitation too!
Simple Western assays have a linear dynamic range of approximately 3 orders of magnitude, letting you detect proteins over a wider concentration range, not to mention with more accurate quantitation.
Simple Western charge-based assays
A Simple Western charge assay is an automated assay just like a Simple Western size assay—without the use of gels, transfer devices, blots, film or manual analysis—but the data you get is very different. Because proteins are separated by the isoelectric point (pI), you'll be able to see extremely small isoelectric point differences. It's a new way of looking at discrete changes in very small samples that you might just be surprised by!
- Peggy Sue and NanoPro 1000 automate traditional 1D isoelectric focusing slab gel protocols including sample loading, charge-based protein separation, immunoprobing, detection and data analysis.
- Low abundance proteins in limited cell populations are a specialty and as few as 25 cells per assay are required.
- Up to 96 samples can be processed at once and assays take just 19 hours to complete with reported pIs and quantitation results.
- You can characterize actions of kinase inhibitor drugs or study on coprotein control mechanisms in primary tissues or fine needle aspirates.
How do Simple Western charge-based assays work?
Simple Western charge-based assays take place in a capillary. Just load your samples and off to the races you go!
Samples and reagents are loaded into an assay plate and placed in Peggy Sue or NanoPro 1000. Proteins and ampholytes are loaded into the capillary automatically and separated by charge and resolve according to their expected pI values. 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. The resulting chemiluminescent signal is detected and quantitated.
What Does Simple Western Charge Data Look Like? Simple Western charge-based assay data is processed automatically in Compass software. Sample data is displayed by electropherogram where it's easier to see minute changes. Quantitative results such as pI value, signal intensity (area), % area, and signal-to-noise for each immunodetected protein are presented in the results table automatically.
Which system is right for you?
|Simple Western System||Jess||Abby||Wes||Sally Sue||Peggy Sue||NanoPro 1000|
|Simple Western size assays|
|Simple Western charge assays|
|Maximum samples per run||25||25||25||96||96||96|
|Run time for max samples (size)||< 3 hours||< 3 hours||< 3 hours||14-19 hours||11-19 hours||N/A|
|Run time for max samples (charge)||N/A||N/A||N/A||N/A||11 hours||11 hours|
|Sample cooling (size)||N/A||N/A||N/A||10°C||10°C||N/A|
|Sample cooling (charge)||N/A||N/A||N/A||N/A||3°C||3°C|
|Operating humidity range||20-60% relative, non-condensing|
|Operating temp range||18-24°C|
|Power||100-230 V AC, 50/60 Hz|
|Dimensions (H x W x D)||33 x 33 x 52 cm||33 x 33 x 52 cm||33 x 33 x 52 cm||84 x 94 x 61 cm||84 x 94 x 61 cm||84 x 94 x 61 cm|
|Immunoassay Size Specification||Immunoassay Charge Specification|
|Sample required||0.3-1.2 µg lysate||2.0-4.0 µg||0.6-1.2 µg|
|Size or pI range||2-40, 12-230 and 66-440 kDa||2-40, 12-230 and 66-440 kDa||Widest gradient ranges
from pI 3 to pI 10
(± percent difference in MW)
|± 15-20% for MW
< 20 kDa
± 10% for MW
> 20 kDa
|± 15-20% for MW
< 20 kDa
± 10% for MW
> 20 kDa
|± 0.1 pI units|
|Dynamic range||up to 4 logs***||4 logs||3 logs|
|Sensitivity||Low pg||Low pg||Low pg|
|Capillary||5 cm, 100 µm, 400 nL|
*inter-assay CV is with system control
**percent peak area
***Jess/Wes with HDR detection mode.