Simple Western systems

Gel-free, blot-free, hands-free

Simple Western™ platforms let you perform automated western blotting. Separate and analyze proteins by size from 2-440 kDa either by immunoassay or total protein analysis. There's even an automated charge-based assay utilizing isoelectric focusing if you're doing post-translational modification characterization. You'll get quantitative results, reproducibility that's spot on, and use less sample in the process. So, what's the big difference between Simple Western automated western blot systems and traditional western blot tests? Our Simple Western instruments are the only gel-free, blot-free, and hands-free capillary-based immunoassay platforms that integrate and automate the entire protein separation and detection process. So you'll have more time to get down to real science.

Automated Western Blotting with Jess for Simple Western

Jess

Size assays on up to 25 samples
Chemiluminescence and Fluorescence

Automated Western Blotting with Wes for Simple Western

Wes

Size assays on up to 25 samples
Chemiluminescence

Automated Western Blotting with Sally Sue for Simple Western

Sally Sue

Size assays on up to 96 samples
Chemiluminescence

Automated Western Blotting with Peggy Sue for Simple Western

Peggy Sue

Size and charge assays on up to 96 samples
Chemiluminescence

Automated Western Blotting with NanoPro1000 for Simple Western

NanoPro 1000

Charge assays on up to 96 samples
Chemiluminescence

 
 
Simple Western Applications

Cancer

Simple Western Instrument Cancer Research Applications
 
 
 
Simple Western Assay: How does it work?

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.

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, Wes, Peggy Sue, or Sally Sue and press start—it's a complete, walk-away solution for protein separation and detection.

  • A Simple Western size assay is an automated western blot—no gels, no transfer devices, no blots, no film and no manual analysis. Just load your samples in Jess, Wes, Peggy Sue, or Sally Sue and press start—it's a complete, walk-away solution for protein separation and detection.
  • Jess, 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, 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.

Immunoassay Workflow Using Automated Western Blotting with Simple Western Technology

Interested in looking at all the proteins in your lysate? Just leave out the antibody and let Jess, 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.

Total Protein Assay Workflow Using Automated Western Blotting with Simple Western Technology

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.

Lane View Data For Simple Western Size-based Assay

Just need total protein? We've got that covered too!

Automated western data for total protein detection

Total Protein detection. Decreasing concentrations of DNAK in Hela lysate and negative controls (15, 7.5 and 3.75 µg/mL).

If you're more familiar with capillary electrophoresis, you can see your results in a more traditional electropherogram view too.

Size based Simple Western electropherogram

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.

Lane View of Automated Western Results From High Molecular Weight Targets

Detection of 3 high molecular weight targets. HECTH9, Talin and UBR5 were detected in K562 lysate using the 66-440 kDa Master Kit.

Reproducibility and quantitation

Simple Western assays are fully automated by Jess, 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!

PI3K expression Results Shown in Lane View From Automated Simple Western Application
PI3K table

Phosphoinositide 3-kinase (PI3K) expression in an MCF-7 lysate. Ten replicates (n=10) of MCF7 lysate at 1 µg/µL were analyzed using a PI3K-specific primary antibody. The predicted molecular weight of PI3K is 110 kDa. Simple Western assay data reported an average molecular weight of 107 kDa with a 0.5% CV for sizing and an 8.7% CV for signal intensity (area).

Dynamic range

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.

Quantitation of GSK-3 alpha with Simple Western

Dynamic range of detection for GSK-3 alpha. Lysate from K562 cells lysed in Bicine/CHAPs buffer was serially diluted and analyzed using a GSK-3 alpha antibody. The calculated dynamic range was 3.3 logs with an R² value of 0.9995.

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.

Simple Western charge-based immunoassay

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.

Charge-based assay data from Simple Western

Simple Western systems comparison chart

Which system is right for you?

Simple Western System Jess Wes Sally Sue Peggy Sue NanoPro 1000
Simple Western size assays  
Simple Western charge assays      
Chemiluminescence
Fluorescence        
Maximum samples per run 25 25 96 96 96
Run time for max samples (size) < 3 hours < 3 hours 14-19 hours 11-19 hours N/A
Run time for max samples (charge) N/A N/A N/A 11 hours 11 hours
Sample cooling (size) N/A N/A 10°C 10°C N/A
Sample cooling (charge) 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 84 x 94 x 61 cm 84 x 94 x 61 cm 84 x 94 x 61 cm
Part number 004-650 004-600 004-700 004-800 004-109

 

  Immunoassay Size Specification Immunoassay Charge Specification
Description Chemiluminescence Fluorescence  
Sample required 0.3-1.2 µg lysate 0.6-1.2 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
Sizing CV <10% <10% <10%
Intra-assay CV <15% <15% <20%
Inter-assay CV <20%* <20%* <20%**
Resolution
(± 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
Quantitation CV <20% <20% <20%
Dynamic range 2-3 logs up to 4 logs*** 3 logs
Sensitivity ng 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.