Stain-Free Imaging Technology

Advantages of Using Stain-Free Technology

Stain-Free Technology Provides More Sensitivity and Better Dynamic Range than Coomassie Stains

Protein visualization data obtained from Stain-Free gels are comparable to those obtained from gels stained with other dyes. In general, the sensitivity of Stain-Free gels when visualizing data is equal to that of Coomassie-stained gels for all proteins. For proteins with higher tryptophan content, Stain-Free gels provide much higher sensitivity than CBB-stained gels (Figure 1).

Figure 1. Comparison of a Stain-Free gel, CBB R-250, and Bio-Safe G-250 stained gel images. Serial 1:2 dilutions of broad range unstained molecular weight standards were separated on a 4–20% Criterion Stain Free Tris-HCI Gel. The gel was imaged with a Stain-Free enabled imager, then stained with Coomassie (CBB R-250 and Bio-Safe G-250) stain and imaged on a densitometer. Arrowhead indicates β-galactosidase.
 

The limit of detection for the Stain-Free gels is 8 to 28 ng, similar to that of silver stains (0.6 to 1.2 ng), while Coomassie R-250 stain can detect protein amounts of at least 35 to 50 ng. Some fluorescent stains can detect proteins at levels below the 1 or 0.5 ng limit. Stain-Free gels have more reproducible data with smaller coefficients of variation compared to Coomassie or silver stains (McDonald et al. 2008; McDonald, 2009).

The linear dynamic range for protein quantitation is defined as the range through which the signal intensity on a blot proportionally increases with the increase in protein load. Ideally, the protein load should fall within the quantitative linear dynamic range of the antibody used for its detection (Taylor and Posch, 2014). Stain-Free gels provide a linear dynamic range between 10 and 80 µg of total protein load from cell or tissue lysates at a higher range of protein load (Figure 2A) and from 20 to 1 µg at a lower range (Figure 2B) (Taylor et al. 2013, Hammond et al. 2013).

Figure 2. Linear dynamic range provided by Stain-Free technology for total protein measurements. A. HeLa cell lysate dilutions from 80–2.5 µg total protein; B. HeLa cell lysate dilutions from 20–1 µg total protein.

Compatibility with Downstream Applications

The Coomassie dye-based method does not allow the same gel to be used for transfer during western blotting or for mass spectrometry. This is a serious drawback when the ultimate goal is the identification or quantitation of proteins in western blotting, especially when large gels and sample volumes are required for separation and visualization. In contrast to Coomassie staining, Stain-Free technology is compatible with most downstream applications, such as western blotting, which helps not only when resources are limited, but also in the appropriate normalization of data for quantitation using total protein normalization (see Normalization of Data in Quantitative Western Blotting below).

Verification of Protein Transfer

The proper transfer of proteins to the membrane is critical to the western blotting process. A common standard practice to verify protein transfer is staining the blot with Ponceau S, a negatively charged stain that binds to all positively charged amino acids in a protein. Ponceau S staining is fast and relatively inexpensive, and the stained membrane can then be used for downstream applications such as western blotting. However, the ephemeral nature of the binding of the dye to the protein causes the intensity of the bands on the membrane to decrease rapidly, making detection and quantitation harder. Other relatively expensive blot stains, such as SYPRO Ruby or amido black, are also used for blot quantitation and require special disposal methods. The time needed to view the stain on the blot also varies with the dye used. Protocols for using Ponceau stain recommend at least 5 minutes of staining followed by 15 minutes of washing steps. SYPRO Ruby Blot Stain is more elaborate, requiring fixation, overnight staining, and destaining.

Verifying protein transfer, both from the gel and on the membrane, using a Stain-Free enabled imager provides significant time savings and cost savings for western blotting (Colella et al. 2012). The observed intensity of the bands does not depend on the duration of staining or destaining, a factor affecting dye-based techniques during visualization and quantitation. Also, the intensity of the band on Stain-Free blots, which is the result of a covalent modification, does not decrease with time. Using the Stain-Free method, protein transfer can be verified in as little as 2 minutes.

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  Gel before transfer

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  Membrane after transfer

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  Gel after transfer

Assessment of protein transfer using a Stain-Free enabled imaging system. Images of the gel before and after transfer and of the membrane after transfer were taken using the Criterion Stain-Free Imager. Serial 1:2 dilutions of hemoglobin (starting quantity, 80 ng), with 1.8 μg of BSA/lane as a carrier (top band), were electrophoretically separated on a 4–20%, 26-well Criterion Stain-Free Gel.

FAQs

Q: Will I need to purchase new buffers and reagents to utilize Stain-Free technology?

A: Stain-Free gels do not use specialized buffers or reagents. Standard SDS-PAGE buffers can be used. However, the technique does require an imager that can visualize the activated gel or membrane. View our Stain-Free Imaging Systems.

Q: As Stain-Free technology utilizes the modification of tryptophan residues, will it work on my protein of interest?​

A: Theoretically, even one tryptophan residue is sufficient for signal activation, and proteins that have as few as two tryptophan residues are readily detected and quantified using Stain-Free technology. For most applications, the requirement of tryptophan residues for visualization is not a concern. Data available from UniProt show that only about 10% of proteins from all organisms lack tryptophan and most of those proteins are less than 10 kD in size. In most organisms, approximately 90% of proteins are above 10 kD in size (in the 10–260 kD range) and possess tryptophan residues (Table 1).​

Table 1. Tryptophan content of the predicted proteomes of several model organisms.*

Limits of detection (LOD) and limits of quantitation (LOQ) of proteins on Criterion Stain-Free Gels and Bio-Safe G-250 Stained Gels. Individual bands from broad range unstained protein standards from four replicate gels were used to determine visual LOD and LOQ. Averaged numbers were used to generate the graph.
 

Q: How does Stain-Free affect the way we do protein research today? ​

A: With an increasing number of errors in quantitation arising due to the use of housekeeping proteins and associated normalization issues, a growing number of journals in the western blotting field have forced authors to withdraw published reports, issue errata, or have their publications rejected (Neill 2009). Journals are now requiring strict adherence to the use of internal controls and are mandating the use of imaging techniques that yield linear signal ranges and report linear dynamic range of signal (see guidelines for reporting life sciences research from Nature Publication Group 2013 and guidelines from Journal of Biological Chemistry). Stain-Free technology fits perfectly in this new world of western blotting as it satisfies all the requirements for carrying out these new protocols and is considered an effective strategy to improve accuracy of western blots (Ghosh et al. 2014).

Meeting the New Publication Requirements for Quantitative Western Blotting

Plasmid Purification Kit Selection Guide

Kit		DNA Yield	Preparation Time	Bacterial Culture Density or Volume	Purification Format	Binding Matrix	Growth Media
Aurum plasmid mini		>20µg	10 min	Up to 12 OD • ml*	Vacuum** and spin	Silica membrane	Standard
Quantum Prep miniprep		>20µg	15 min	1–10 ml	Spin	Diatomaceous earth	Enriched or standard

 

* (OD₆₀₀ of undiluted culture) x (culture volume in ml) = #of OD • ml. 1 OD₆₀₀ is equivalent to approximately 8 x 10⁸ cells/ml.
** Using the Aurum vacuum manifold.

Resources

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  Total Protein Normalization

  Learn how total protein normalization is key to obtaining quantitative western blot data for publication.

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  Fluorescent Western Blotting

  See how fluorescent western blotting addresses the need for accurate, quantitative determination of protein expression.

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  Film vs. Digital Western Blot Imaging

  Discover how modern digital imaging systems, with wide dynamic range enable, more accurate quantitation over a range of signal intensities that are equal to or better than film.

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  Image Lab Touch Software Resources

  Access Image Lab Touch Software resources such as: instructional videos, information on Smart Tray Technology, and tips on capturing images including exporting data.

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  Image Lab Software Resources

  Bio-Rad provides tutorials for basic acquisition and advanced analysis use of Image Lab Software 6.0.1. Topics include, densitometric analysis, molecular weight, normalization, purity assessment, and more!

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  Crucial Factors of Successful Western Blotting

  Join our western blotting expert and guest speakers for a webinar series exploring factors that go into the design and execution of successful, repeatable western blots. Chemiluminescence or fluorescence, qualitative or quantitative blot — we’ll discuss how-to's and take a deep dive exploring the why's.

Documents Library

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  ChemiDoc MP System Brochure
  (PDF 1.26 MB)

  Explore the features and benefits of the new high-end imaging system for the best fluorescence and chemiluminescence detection.

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  Stain-Free Approach to Western Blotting
  (PDF 864 KB)

  Learn how to use Stain-Free technology for total protein normalization as an alternative to the standard blot normalization process.

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  Stain-Free Western Workflow Brochure
  (PDF 593 KB)

  See how this improved workflow incorporating Stain-Free gel technology saves time and increases the accuracy and reliability of your western blot results.

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  Determining the Appropriate Sample Load When Using a Stain-Free V3 Western Workflow
  (PDF 182 KB)

  Determine the right load for the detection of target proteins before the actual western blot experiment is carried out.

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  Validating the Expression Consistency of a Housekeeping Protein
  (PDF 198 KB)

  Validate the consistency of HPK expression before the actual western blot experiment is performed.