Stain-Free Western Blotting

Bio-Rad Stain-Free western blotting is a streamlined approach to getting better western blotting results. Stain-Free technology adds speed and enables quality assessment at each step of a western blotting experiment — from running gels to quantitating proteins. In addition to the ability for total protein normalization (TPN) without the need to perform extra staining steps; this technology empowers researchers to achieve faster results and better data.

Stain Free Enabled

Stain-Free technology is superior to conventional western blotting methods as it allows users to:

  • Visualize gels minutes after electrophoresis
  • Perform quality assessments at each step without the need for additional staining and destaining
  • Increase accuracy and produce reliable quantitative results via total protein normalization
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Stain-Free Western Blotting Workflow

1
Mini Protean Tetra Cell for Western Blotting
15 minutes

Separate Proteins

Speed with flexibility: TGX Stain-Free Gel chemistry available in precast and handcast formats

Mini Protean Tetra Cell
Mini Protean TGX Stain-Free Gels

2
Visualize Protein Separation
Visualize Protein Separation

Pre-transfer gel image

1 minute

Visualize Protein Separation

Coomassie-like performance with no background variability and no staining/destaining

Stain-Free Enabled Imagers

3
Protein Transfer
Visualize Protein Separation

Post-transfer gel image

3 minutes

Protein Transfer

  • Fast, efficient, and uniform protein transfer
  • Throughput: transfer 4 mini or 2 midi gels at once
  • Convenience: choose from ready-to-use transfer packs (nitrocellulose or PVDF)

Trans-Blot Turbo Transfer System

4
Visualize Protein Separation
Visualize Protein Separation

Blot gel image

1 minute

Assess Transfer Efficiency

Quickly verify quality of transfer by imaging gel post transfer

Stain-Free Enabled Imagers

Faster Blocking, Antibody Incubation, and Blot Detection Workflow:

A workflow that enables five-minute blocking, antibody incubation, and blot detection within approximately 4 hours — faster than traditional methods!

EveryBlot Blocking Buffer

EveryBlot Blocking Buffer

Enables 5-minute blocking and maximum sensitivity for all western blots.

PrecisionAb Validated Western Blotting Antibodies

PrecisionAb Validated Western Blotting Antibodies

Highly specific and sensitive primary antibodies that have been extensively validated for western blotting.

StarBright Blue Fluorescent Secondary Antibody

StarBright Blue Fluorescent Secondary Antibody

Ultra-sensitive fluorescence detection with very low background of single or multiple proteins in one blot without stripping or reprobing.

 
5

Normalize Total Protein and Analyze Western Blot Data

Easy multiplexing
Use western blot validated, highly specific PrecisionAb Primary Antibodies and our StarBright Blue Fluorescent Secondary Antibodies to detect multiple targets on the same blot.

Image Lab Software
Visualize Protein Separation

Total protein detection with chemiluminescence

Visualize Protein Separation

Total protein detection with fluorescence

Visualize Protein Separation

Stain-free blot (from step 4) for normalization

Benefits of using Stain-Free blot image as total protein loading control

  • Eliminates the need to strip then reprobe, and cut the blot
  • More precise normalization methods compared to traditional methods which use a single housekeeping protein
  • Produces reliable and accurate quantitation

Image Lab Software

Total Protein Normalization

Screenshot of Image Lab software

An illustration of the total protein normalization feature in the Image Lab Software using a Stain-Free enabled imager.

Bio-Rad’s Stain-Free western blotting provides a practical, convenient, and reliable way to perform total protein normalization. Stain-Free labeled proteins can be visualized in gels 1 minute after gel electrophoresis. Stain-Free blot visualization and image acquisition require only a few seconds using a stain-free enabled imager. This technology makes it easy for researchers to adopt total protein loading control for western blotting.

Total protein normalization using Stain-Free technology

  • Enables total protein in load sample to be measured
  • Eliminates immuno-blotting interference associated with HKP
  • Accounts for variations during loading, electrophoresis, and transfer

Why is Stain-Free Total Protein Normalization Superior over Other Methods?​

The use of traditional total protein stains can hinder downstream visualization of a western blot, usually requiring repetitive staining and detaining steps which is one of the obstacles preventing wider adaptation of TPN. The unique characteristics of Stain-Free technology makes imaging of total protein a much better method for quantitative western blotting.

  • No UV

     
  • With UV

    Gel Without Stain-Free TechnologyGel Without Stain-Free Technology

Following a brief UV light activation, Stain-Free fluorochromes are covalently bound to protein molecules in the gel, allowing them to be imaged repeatedly on the gel or on a membrane post-transfer.

Total Protein normalization using Stain-Free technology consistently delivers scalar linear response across a wider dynamic range.

Stain-Free Total Protein Normalization for Western Blotting

Linearity comparison of stain-free total protein measurement and immunodetection of three housekeeping proteins in 10–50 μg of HeLa cell lysate. On the left are representative images of (a), stain-free blot and the chemi blots for (b), β-actin; (c), β-tubulin and (d), GAPDH. Lane labels correspond to total protein load (μg). Although the actin and tubulin signals appear linear, the densitometric ratio was far below the predicted “quantitative response” of actual loading whereas the stain-free signal correlated to the expected result (e).

Reference
Taylor SC Posch A (2014). The design of a quantitative western blot experiment. Biomed Res Int. 36, 1590

 

Stain-Free technology allows scientists to visualize strong total protein signals with low background on their gels and blots that would otherwise not be possible without additional staining steps thus, improving overall dynamic range without the need to perform extra staining and destaining steps.

  • With Stain-Free Technology

    Gel With Stain-Free Technology
  • Without Stain-Free Technology

    Gel Without Stain-Free Technology

Comparison between high background and low background signal in gels. Stain-Free technology noticeably lowers the background signal in comparison to traditional total protein detection methods.

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Ordering

  • Mini-PROTEAN Tetra Vertical Electrophoresis Cell for Mini Precast Gels, 4-gel #1658004

    Mini-PROTEAN Tetra Vertical Electrophoresis Cell for Mini Precast Gels, 4-gel #1658004

    4-gel vertical electrophoresis system, includes electrode assembly, companion running module, tank, lid with power cables, mini cell buffer dam

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  • Criterion Cell #1656001

    Criterion Cell #1656001

    Vertical midi-format electrophoresis cell, includes buffer tank, lid with power cables, 3 sample loading guides (12+2-well, 18-well, and 26-well)

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  • Trans-Blot Turbo Transfer System #1704150

    Trans-Blot Turbo Transfer System #1704150

    Blotting instrument, includes base, 2 cassettes to hold 1–2 midi or up to 4 mini blotting sandwiches, blot roller

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  • ChemiDoc Imaging System #12003153

    ChemiDoc Imaging System #12003153

    The ChemiDoc Imaging System allows sensitive imaging and documentation of DNA/protein gels and chemiluminescent western blots

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  • ChemiDoc MP Imaging System #12003154

    ChemiDoc MP Imaging System #12003154

    The ChemiDoc MP Imaging System is a full-feature, best-in-class instrument for imaging and quantifying nucleic acid and protein in gels and western blots. It is capable of imaging fluorescent western blots (RGB, far-red, near-IR), chemiluminescent western blots, all common nucleic acid and protein gel stains, plus stain-free gels. Includes internal computer, 12" touch-screen display, camera, Image Lab Touch Software, and Image Lab Software

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  • Mini-PROTEAN TGX Stain-Free Precast Gels

    Mini-PROTEAN TGX Stain-Free Precast Gels

    Based on the long-shelf-life TGX (Tris-Glycine eXtended) formulation and include unique trihalo compounds that allow rapid fluorescent detection of proteins with the ChemiDoc MP and GelDoc Go Imaging Systems.

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  • Criterion TGX Stain-Free Precast Gels

    Criterion TGX Stain-Free Precast Gels

    Based on the long shelf life TGX formulation and include unique trihalo compounds that allow rapid fluorescent detection of proteins with the ChemiDoc MP and GelDoc Go Imaging Systems.

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Applications & Technologies

  • Stain-Free Imaging Technology

    Stain-Free Imaging Technology

    Visualize proteins on gels and blots without staining and destaining steps, and use total protein normalization for quantitative western blots.

  • Western Blotting Techniques

    Western Blotting Techniques

    Learn more about western blotting techniques. Find step-by-step protocols and helpful tips on equipment, membranes, transfer conditions, and detection methods.

  • Protein Separation and Analysis

    Protein Separation and Analysis

    Bio-Rad's Stain-Free Western Blotting Workflow facilitates speed and validation at each step of a western blotting experiment — from running gels to quantifying proteins.

  • Protein Electrophoresis

    Protein Electrophoresis

    Find protocols, video tutorials, and selection guides to help you at every step of your electrophoresis experiments.

  • Imaging and Analysis

    Imaging and Analysis

    Find information on protein visualization and quantitation methods, gel and blot imaging instrumentation, and image analysis software.

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