Western Blotting

The Complete Guide to
Stain-Free Western Blotting

Visit bio-rad.com/Stain-Free for more information.

Sample Prep

Electrophoresis

Transfer

Immunodetection

Image Acquisition

Image Analysis

WelcomeIn this eBook, you will learn about the benefits of the proprietary Stain-Free technology from Bio-Rad
Laboratories, Inc., get guidance for each step of the workflow, and discover Bio-Rad resources to ensure
you are efficient and successful in western blotting.

Table of Contents
The Bio-Rad Western Blotting Workflow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  3
What Is Stain-Free Imaging Technology?. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  3
Stain-Free Workflow Compared to Traditional Western Blotting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  4
The Advantages of Stain-Free Technology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  5
The Dynamic Range of Stain-Free Technology. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  6
Verify Your Protein Transfer in Minutes with No Staining. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  6
Stain-Free Technology Is Compatible with Downstream Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  7
Summary of Stain-Free Technology Advantages. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  7
The Stain-Free Western Blotting Workflow. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  8
Sample Preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  8
Electrophoresis, Activation, and Imaging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  8
Transfer. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  8
Immunodetection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  9
Image Acquisition. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  9
Image Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  9
Meet the Bio-Rad Western Blotting Imagers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Benefits of Total Protein Normalization (TPN) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
The Stain-Free Western Blotting Workflow Meets New Publication Guidelines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
Considerations When Using Stain-Free Technology. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
Conclusion and Resources . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

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Sample Prep

Electrophoresis

Transfer

Immunodetection

Image Acquisition

Image Analysis

The Bio-Rad Western Blotting Workflow
Western blotting is a powerful technique to separate and detect proteins, estimate their quantities, and determine their molecularweights. Proteins of interest are extracted from cells or tissues and processed in a multistep western blotting workflow. Western blots
can also be used in a variety of other applications, such as characterization of protein-protein interactions, posttranslational modifications,and antibodies.
The Bio-Rad Western Blotting Workflow consists of six steps, each critical to producing high-quality data:
1.

Sample preparation

2.

Electrophoresis

3.

Transfer

4.

Immunodetection

5.

Image acquisition

6.

Image analysis

In this guide, you will learn how Stain-Free technology fits into the western blotting workflow, and how this technology can improveyour western blotting workflow and results.

What Is Stain-Free Imaging Technology?
Stain-Free technology uses a polyacrylamide gel containing a proprietary trihalo compound that makes proteins fluoresce directly in thegel after a short ultraviolet (UV) photoactivation (Figure 1). This trihalo compound covalently binds to tryptophan residues, enhancing theirfluorescence when exposed to UV light. As a result, you can immediately visualize proteins in gels or on blots. Stain-Free technology
enables the detection of proteins at levels as low as 10–25 ng. A Bio-Rad Stain-Free–enabled imager is required.

UV activation(5 min)Run gel as usual. Fluorophore does not affectband migration.

Ready forvisualization

The fluorophore binds covalently (permanently) toproteins during the activation step.

The gel can be imaged immediately, or run further.
Proteins can also be imaged on the blot after transfer.

Fig. 1. Stain-Free gel activation. Activate Stain-Free gels with UV light and visualize total protein in minutes.

BIO-RAD LABORATORIES, INC. | 3

Sample Prep

Electrophoresis

Transfer

Immunodetection

Image Acquisition

Image Analysis

Stain-Free Workflow Compared to Traditional Western Blotting
The Stain-Free workflow takes only 5 hours to complete, versus 16 hours for traditional western blotting. In addition to saving time,
Stain-Free technology also allows you to assess transfer efficiency and collect other information quickly and easily during
the western blotting process.
Stain-Free Western Blotting

Advantages of
the Stain-Free Workflow

Sample Preparation

Traditional Western Blotting
Sample Preparation–

Electrophoresis and Imaging

Electrophoresis and Imaging
TGX Stain-Free Gels
can be run in as little
as 16 minutes

Transfer and Imaging

Transfer and Imaging
Trans-Blot™ Turbo Transfer
System transfers in 7 min
No stain necessary
Verify transfer immediately

Immunodetection

Immunodetection
Verify transfer before
adding antibody,saving time and cost

Image Acquisition

Image Acquisition–

Image Analysis

Image Analysis
TPN signal is a goodloading control —no stripping andreprobing needed

4 | BIO-RAD LABORATORIES, INC.

Sample Prep

Electrophoresis

Transfer

Immunodetection

Image Acquisition

Image Analysis

The Advantages of Stain-Free Technology
Stain-Free Technology Provides More Sensitivity than Coomassie Blue Stains
Stain-Free gel data are comparable to data from gels stained with traditional dyes, with a few exceptions. In general, the sensitivity of
Stain-Free technology is equal to that of Coomassie Brilliant Blue (CBB) for proteins containing low-to-medium levels of tryptophan.
However, for proteins with higher tryptophan content, Stain-Free technology provides much higher sensitivity than CBB (Figure 2).
Stain-Free Technology
1

2

3

4

5

6

CBB R-250 Stain
7

8

1

2

3

4

5

6

Bio-Safe Coomassie Stain
7

8

1

2

3

4

5

6

7

8

9

10

Fig. 2. Comparison of a Stain-Free gel image with Coomassie Brilliant Blue (CBB) R-250– and Bio-Safe Coomassie (G-250)–stained gel images. Serial 1:2 dilutionsof 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 stains and imaged on a densitometer. Arrow indicates ß-galactosidase.

The limit of detection for the Stain-Free gels is 8–28 ng, similar to that of silver stains (0.6–1.2 ng), while Coomassie Brilliant Blue R-250
Staining Solution can detect protein amounts of at least 35–50 ng. Some fluorescent stains detect proteins below the 1 or 0.5 ng limit.
Stain-Free gels have more reproducible data with smaller coefficients of variation compared to CBB or silver stains (McDonald et al. 2008,
McDonald 2009).
Visit bio-rad.com/Stain-Free to learn more about Stain-Free technology.

BIO-RAD LABORATORIES, INC. | 5

Sample Prep

Electrophoresis

Transfer

Immunodetection

Image Acquisition

Image Analysis

The Dynamic Range of Stain-Free Technology
The linear dynamic range for protein quantitation is the range where the signal intensity on a blot proportionally increases with the increasein 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 predictable linear dynamic range of 10–80 µg of total protein load from cell or tissue lysates at ahigher range of protein load (Figure 3A) and from 1–20 µg at a lower range (Figure 3B) (Taylor et al. 2013, Hammond et al. 2013).
8,000,000
Signal intensity

7,000,000
6,000,000

B

3,000,000

y = 88887x – 39752
R2 = 0.9997

5,000,000
4,000,000
3,000,000
2,000,000

y = 109285x + 254001
R2 = 0.9847

2,500,000
Signal intensityA

2,000,000
1,500,000
1,000,000
500,000

1,000,000
0
10 20 30 40 50 60 70 80 90
Total protein concentration, µg
In-gel standard deviation: ~10%
Gel-to-gel standard deviation: ~15%

0
0

5
10
15
20
25
Total protein concentration, µg

In-gel standard deviation: ~10%
Gel-to-gel standard deviation: ~15%

Fig. 3. Linear dynamic range provided by Stain-Free technology for total protein measurements. A, HeLa cell lysate dilutions from 80 to 2.5 µg total protein; B, HeLa cell lysate
dilutions from 20 to 1 µg total protein.

Verify Your Protein Transfer in Minutes with No Staining
The complete transfer of proteins to the membrane is critical in western blotting. Traditional verification methods like Ponceau S, SYPRORuby, and other blot stains are more time-consuming than the Stain-Free method. Verifying protein transfer from gels to membranes usinga Stain-Free–enabled imager can be accomplished in as little as 2 minutes. Unlike with traditional dye-based techniques, the observed
intensity of the bands does not depend on the duration of staining or destaining (Figure 4).
A. Gel before transfer

B. Membrane after transfer

C. Gel after transfer

Fig. 4. Rapidly assess transfer using Stain-Free technology. Images were taken of the gel before (A) and after (B) transfer and of the membrane after transfer (C) using theCriterion Stain-Free Gel Imaging System. Serial 1:2 dilutions of hemoglobin (starting quantity, 80 ng), with 1.8 μg of bovine serum albumin (BSA)/lane as a carrier (top band),were electrophoretically separated on a 4–20% Criterion Stain-Free Tris-HCl Gel, 26-Well.

6 | BIO-RAD LABORATORIES, INC.

Sample Prep

Electrophoresis

Transfer

Immunodetection

Image Acquisition

Image Analysis

Stain-Free Technology Is Compatible with
Downstream Applications
Proteins that have passed through the Stain-Free workflow are compatible with many downstream applications such as western
blotting, mass spectrometry, and chromatography (Figure 5). The Coomassie dye–based method does not allow the same gel to be
used for transfer during western blotting or for mass spectrometry. This compatibility streamlines the workflow and enables total protein
normalization and quantitation.
Stain-Free Technology

Western Blotting
Increase confidence with rapid
checkpoints for separationand transfer and get accuratequantitation using total protein
normalization.

Chromatography
Quickly verify protein
fraction purity during method
development and routine
chromatography without
any messy, time-consuming
staining steps.

Mass Spectrometry
Improve ease of processing
and time to results by removing
manual staining and destaining
steps and allowing for quick
visualization of gels.

Fig. 5. Compatibility of Stain-Free technology with common downstream applications.

Summary of Stain-Free Technology Advantages■■■

 ffers more sensitivity and better dynamic range thanO
Coomassie Blue stains

Watch a video about the benefits of Stain-Free technology:

 erforms quality assessments at each step without the needPfor staining and destainingIncreases accuracy and produces reliable quantitative results
via total protein normalization■

Is compatible with downstream applications■

Visualizes gels minutes after electrophoresis

BIO-RAD LABORATORIES, INC. | 7

Sample Prep

Electrophoresis

Transfer

Immunodetection

Image Acquisition

Image Analysis

The Stain-Free Western Blotting Workflow
Sample Preparation
Maintain the same sample preparation workflow with Stain-Free imaging technology.
Use a protein quantitation assay to determine the concentration of your sample.

Electrophoresis, Activation, and Imaging
Immediately activate your gel to check transfer efficiency with Stain-Free technology.
Electrophoresis is performed the same way as withstandard western blotting techniques. The main differenceis that after running the gel, you can activate and image it
immediately on a Bio-Rad Stain-Free–compatible imager(Figure 6). If proceeding to western transfer, activate thegel for 45 sec prior to imaging. If gel evaluation is thedesired endpoint, activate for 5 min prior to imaging.

45 sec–5 min

Fig. 6. Example Stain-Free gel image. Gel activation can be performedin as little as 45 seconds, allowing you to perform a quick check prior towestern transfer.

Transfer
After activating your Stain-Free gel, transfer proteins to a membrane following your typical western blotting
transfer protocol.
Once the transfer is complete, capture another gel image to evaluate the transfer process. When imaging, ensure afair comparison by using the same exposure time and contrast settings you used to image the gel prior to transfer(Figure 7). To finish evaluating the transfer, image the Stain-Free blot and check all three images. If the transfer iscomplete, then proceed to immunodetection.

1–5 sec

Post-run Stain-Free gel

1–10 sec

Membrane after western transfer

1–5 sec

Stain-Free gel after western transfer

Fig. 7. Stain-Free technology makes it easy to check that western transfer is complete. Left, pre-transfer gel image; center, post-transfer membrane
image; right, post-transfer gel image. Because activation is nonreversible, it does not need to be performed again after western transfer. Gels andmembranes can be imaged in seconds.

BIO-RAD LABORATORIES, INC. | 8

Sample Prep

Electrophoresis

Transfer

Immunodetection

Image Acquisition

Image Analysis

Immunodetection
No change to your immunodetection workflow is necessary with Stain-Free imaging technology.
Both chemiluminescence and fluorescence detection methods are compatible.

Image Acquisition
Once your blot is ready for image acquisition, use a Bio-Rad Stain-Free–enabled imager to acquire images of theStain-Free blot.
Stain-Free Gel Imaging of Chemiluminescence Western Blots

For chemiluminescence detection, select Blots in Applications, then the Chemi option. Acquire a blot image on the
Stain-Free–enabled imager just before applying the enhanced chemiluminescence (ECL) reagent to achieve the mostaccurate representation of the total protein present (Figure 8).

Electrophoresis
Stain-Free blot

Transfer

Immunodetection

Chemiluminescence blot

Fig. 8. Examples of Stain-Free and chemiluminescence blot images.

Image Acquisition
Image Analysis

Stain-Free Imaging of Fluorescence Western Blots

For multiplex western blot applications, use the red, far red (FR), and near infrared (NIR) channels for detecting proteins ofinterest and reserve the blue channel for total protein detection using Stain-Free technology (Figure 9).

Fig. 9. Examples of multiplex western blots using 800 nm, 650 nm, and rhodamine channels.

Image Analysis
Perform image analysis for a Stain-Free blot the same way you would for traditional western blotting using
Image Lab Software.
Volume Box Tools and Lane and Band Tools are two common approaches for quantitating western blots.
Use Total Protein Normalization to equalize your quantitative western blot.

BIO-RAD LABORATORIES, INC. | 9

Sample Prep

Electrophoresis

Transfer

Immunodetection

Image Acquisition

Image Analysis

Meet the Bio-Rad Western Blotting Imagers

ChemiDoc MP Imaging System

ChemiDoc Go Imaging System

ChemiDoc Imaging System

GelDoc Go Gel Imaging System

High-end imaging system for the best
fluorescence and chemiluminescence
detection

Advanced complementary metaloxide semiconductor (CMOS) digital
imaging allows chemiluminescence,colorimetric, or fluorescence
detection when imaging StarBright™
Blue Fluorescent Secondary
Antibodies

Chemiluminescence sensitivity
matching X-ray film

Best for smaller research labs doingroutine gel documentation■

 igh-sensitivity detection of secondaryH
antibodies in red, green, and blue (RGB)and FR/NIR channels with minimal
background fluorescence ompatible with StarBright Blue 520
C
and StarBright Blue 700

 hemiluminescence sensitivity
Cmatching X-ray film■

 asy-to-use software, perfect forE
multi-group use■

Multiplex fluorescence detection■

Chemiluminescence sensitivity■

Multiplex fluorescence detection■

Easy-to-use instrument interface■

 hemiDoc MP Installation/Operational
C
Qualification (IQ/OQ) Kit available■

Intuitive software, dedicated printersupport, BR.io cloud storage, and WiFi
and Ethernet connection capabilities■

ChemiDoc Go IQ/OQ Kit available

■

■

■

■

■

 asy-to-use software perfect forE
multi-group use pgradeable to ChemiDoc MP
U
System for multiplex fluorescence
detection
ChemiDoc IQ/OQ Kit available■

High-resolution personal imaging■

 asy-to-use software with minimalE
setup time■ arge imaging area with aLcompact footprint■IQ/OQ tools and software formanaging user accounts andpermissions

Visit bio-rad.com/ChemiDoc to learn more about ChemiDoc Systems.
Visit bio-rad.com/ChemiDocGo to learn more about the ChemiDoc Go System.
Visit bio-rad.com/GelDocGo to learn more about the GelDoc Go System.

Analyze with Certainty on a PC or Mac

Automated lane and band detection, molecular weight determination, and normalization are just a few clicks awaywith Image Lab Software.

Annotation to make interpretation andpublication easy.

Stain-Free imaging allows for total
protein normalization.

Lane profiling of total protein signal helps remove backgroundand refine band detection.

Visit bio-rad.com/ImageLab to learn more about Image Lab Software.

BIO-RAD LABORATORIES, INC. | 10

Sample Prep

Electrophoresis

Transfer

Immunodetection

Image Acquisition

Image Analysis

Benefits of Total Protein Normalization (TPN)
Reliable assessment of changes in target protein expression levels requires measuring both the target protein and the loading control
protein in their linear dynamic ranges for immunodetection. Traditionally, housekeeping genes like actin, b-tubulin, or GAPDH have served
as loading controls and in data normalization (Figure 10). Unfortunately, housekeeping proteins (HKPs) are usually highly expressed,
whereas target proteins are often expressed in low abundance. In the HKP approach, large amounts of cell lysates may need to be loaded
to detect target proteins, which can result in the overloading of HKPs and yield oversaturated reference bands out of their linear range.

50

40

30

20

10

B. b-actin
50

40

E. Stain-Free total protein vs. housekeeping proteins (HKPs)
30

20

C. b-tubulin
50

40

30

20

10

D. GAPDH
50

40

30

20

6

10

10

Relative intensity of protein bands

A. Stain-Free blot image

5
4
3
2
1
0
0

10

20
30
40
HeLa cell lysate, µg5060Fig. 10. Stain-Free assessment of several housekeeping proteins provides a linearity comparison of Stain-Free total protein measurement and immunodetection of three HKPs in 10–50 µg
of HeLa cell lysate. A–D, representative images of a Stain-Free blot (A) and chemiluminescence blots for b-actin (B), b-tubulin (C), and GAPDH (D). Lane labels correspond to total proteinload in µg. E, relative signal intensity for total protein (assessed via Stain-Free imaging) and housekeeping proteins. GAPDH ( ) signal intensity remains relatively constant over assessed
concentrations. Although the b-actin () and b-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 ().

Furthermore, HKP expression levels may not be constant but instead may vary with different experimental treatments and other factors.
Stain-Free technology eliminates these issues by quantifying total proteins on the same blot as the proteins of interest. This method ofdata normalization, called total protein normalization (TPN), provides more accurate protein quantitation and western blotting results than
traditional HKP approaches.
In this method, the total density for each lane is measured from a blot, and a lane profile is obtained. Specialized software from
Stain-Free–enabled imagers can interpret the data from the lanes in three dimensions so that the same lane profile data can be viewed
as a three-dimensional peak. The background is adjusted in such a way that the total background is subtracted from the sum of thedensity of all the bands in each lane.

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Sample Prep

Electrophoresis

Transfer

Immunodetection

Image Acquisition

Image Analysis

How to Apply TPN: Step by Step
1■

Treatment 1

Reference

Treatment 2

Protein ofInterest

2■■

Reference

Treatment 1

Reference

Treatment 1

Treatment 2■

Protein ofInterest

3■

Treatment 2

 uantify the signals from the protein of interest and the total
Q
protein signal in each lane se software packages such as Image Lab Software toUaccess tools for TPN

Choose a reference lane for the blot
 se any lane as the reference, since the normalization factorUwill be applied to all other lanes as the ratio relative to thereference lane or each lane, determine the normalization factor by dividing theFsignal intensity of the reference lane by total protein of the sample

Protein ofInterest

Nf = 2

Normalization
factor (Nf )Nf = 0.5

Nf =4

Total protein from reference sample
Total protein from treated sample■

Reference

Treatment 1
x2

Protein ofInterest

Treatment 2
x 0.5

■

 pply the normalization factor to the protein of interest in eachAlane by multiplying the protein signal by the normalization factor
for that lane ow you have the normalized signal for your protein of interest,Nand the relative expression of the proteins can be accurately
compared between lanes

Normalized value = N × band quantity

BIO-RAD LABORATORIES, INC. | 12

Sample Prep

Electrophoresis

Transfer

Immunodetection

Image Acquisition

Image Analysis

The Stain-Free Western Blotting Workflow
Meets New Publication Guidelines
Several major academic publishers have recently revised their editorial guidelines for data publication, emphasizing reproducibility andquantitation criteria. For example, the Journal of Biological Chemistry (JBC) provides guidelines for collecting and presenting data thatspecifically address quantitative western blot publication, guidance on normalization methods and supporting data, and concerns
regarding HKP in protein normalization (see Table 1).
Table 1. Journal of Biological Chemistry (JBC) guidelines for publication and their compatibility with Bio-Rad western blotting workflow solutions.

JBC Guidelines: Quantitative Blots

How to Meet Requirements

Bio-Rad Western Blotting Workflow Solutions“Housekeeping proteins should not be used
for normalization without evidence thatexperimental manipulations do not affect theirexpression”

Use TPN instead of HKPsThe Stain-Free Western Blotting Workflow suite simplifies
Stain-Free TPN. This technology is proven with over
100 publications, allows for improved accuracy, andwill make your data more reliable“Methods including detection of enhanced
chemiluminescence using X-ray film have avery limited dynamic range”

Use an imaging system with at least
4 logs of dynamic range

ChemiDoc Imaging Systems are Stain-Free imagers withthe sensitivity of film. Image Lab Software enables simple
image generation and quantitation“A description of the data supporting thespecificity of all antibodies is required”

Use fully validated antibodies

PrecisionAb™ Antibodies offer documented specificity
and validation protocols

Further Reading
Nature Portfolio. Image integrity and standards. https://www.nature.com/nature-portfolio/editorial-policies/image-integrity,
accessed April 26, 2024.
Fosang AJ and Colbran RJ (2015). Transparency is the key to quality. J Biol Chem 290, 29,692–29,694.
McNutt M (2014). Journals unite for reproducibility. Science 346, 679.
Yadav G and Oh K (2018). Defining the new normal in quantitative western blot data. https://www.bioradiations.com/defining-the-newnormal-in-quantitative-western-blot-data, accessed April 26, 2024.

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Sample Prep

Electrophoresis

Transfer

Immunodetection

Image Acquisition

Image Analysis

Considerations When Using Stain-Free Technology

LOD

Aprotinin

Lysozyme

Lower limit of quantitation, ng

0

Trypsin inhibitor

0

Carbonic anhydrase

1

BSA

1

Ovalbumin

2

Phosphorylase B

2

Myosin3b-galactosidase

3

Aprotinin

4

Lysozyme

4Trypsin inhibitor

5

Carbonic anhydrase

5

BSA

6

Ovalbumin

6

Phosphorylase B

7

Myosin7b-galactosidase

Lower limit of detection, ng

Because Stain-Free technology requires the modification of tryptophan residues, one potential concern is the ability to apply thistechnology to all proteins. Therefore, before deploying Stain-Free technology, determine whether your protein or proteins of interest
contain tryptophan residues. Proteins that lack tryptophan residues, such as the drug aprotinin, are not detected using this technology(Figure 11). Theoretically, even one tryptophan residue is sufficient for signal activation, and proteins with as few as two tryptophan
residues are readily detected and quantified using Stain-Free technology.

Fig. 11. Limit of detection (LOD) and limit of quantitation (LOQ) of proteinson Criterion TGX Stain-Free Precast Gels ( n ) and Bio-Safe Coomassie(G-250)–stained gels ( n ). Individual protein bands from broad range unstained
standards from four replicate gels were used to determine visual LOD and LOQ.
Averaged numbers were used to generate the graph. BSA, bovine serum albumin.

LOQ

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 (https://www.
ebi.ac.uk/uniprot/index). In most organisms, approximately 90% of proteins are greater than 10 kD in size (in the 10–260 kD range) andpossess tryptophan residues (Table 2).
Table 2. Tryptophan content of the predicted proteomes of several model organisms.*

Species

Total Number ofProteins

Number of Proteins
Lacking Tryptophan

Percentage of Total
Proteins Lacking
Tryptophan

Number of Proteins>10 kD

Number of Proteins>10 kD Lacking
Tryptophan

Percentage ofProteins >10 kD
Lacking Tryptophan

Homo sapiens

40,827

4,209

10.31

37,548

2,754

7.33

Escherichia coli O1:K1/APEC 4,865

458

9.41

4,754

408

8.58

Escherichia coli (strain K12)

4,181

456

10.91

3,879

325

8.38

Escherichia coli O6:K15:H31 4,604

562

12.21

4,147

365

8.80

Rattus norvegicus

12,022

1,081

8.99

11,421745

6.52

Mus musculus

35,344

3,435

9.72

33,262

2,480

7.46

Saccharomyces cerevisiae

5,815

648

11.14

5,563

491

8.83

* Data obtained from UniProt in 2014.

BIO-RAD LABORATORIES, INC. | 14

Sample Prep

Electrophoresis

Transfer

Immunodetection

Image Acquisition

Image Analysis

Conclusion and Resources
You have now finished The Complete Guide to Stain-Free Western Blotting. We have discussed
how Stain-Free technology fits in a traditional western blotting workflow and offers some key
advantages. Stain-Free technology allows you to visualize gels without staining and verify transfer
efficiency. It also provides more sensitivity and dynamic range than Coomassie stains andis compatible with downstream applications. Not only does Stain-Free technology save timeand labor, it also allows you to perform total protein normalization, which is more accurate andpreferred over normalization with HKP. See the resources below to learn more about specific
Stain-Free products and our general western blotting resources. Thank you for your interest,
and we hope you consider switching to Stain-Free technology.

Additional Resources

Western Blotting Protocol:
Mini-PROTEAN TGX Stain-Free Gel Run on the Trans-Blot
Turbo Transfer System Using EveryBlot Blocking Buffer
and Clarity and Clarity Max Western ECL Substrates

New to western blotting? Visit ourWestern Blot Learning Center.

Interested in an immersive
experience? Enroll in our Western
Blotting University courses.

Visit our Western Blotting Protocols
Library for detailed custom Stain-Free
western blotting protocols.

Check out the Stain-Free Western
Blotting Workflow Quick Order Guide.

Check out our Western Blotting
webinar series.

Find the right products for you with
the Western Blotting Selector Tool.

ProtocolWestern Blotting

Bulletin 7442

Introduction
This easy protocol includes acceptable ranges for each applicable step. This protocol can be printed and used at thebench as a checklist for tracking the actual conditions used.

Low End of Range

Ideal Conditions

Add 4x or 2x Laemmli buffer to the sample for a final concentration of 1x. Prepare 50% more than you intend to load.
Heat at 95°C for 5 min.

70
2

3

Centrifuge at 10,000 x g for 5 min.

5,000
2

4

Insert the gel and fill the gel apparatus to fill line (550 ml for one to two gels and 800 ml for three to four gels) with running buffer.

5

Load 10 µl protein ladder/molecular weight standard.

6

Load the gel with desired samples.

7

Run the gel at 300 V until the dye reaches the bottomof the gel.

8

Remove the gel from the cassette and place on the imaging tray.

9

Activate the Stain-Free dye and acquire a gel image.

Transfer✔ StepInstructions

Actual Conditions

Assemble the gel and membrane inside the Trans-Blot Turbo Transfer System cassette.

2

Run the transfer at 2.5 A for 7 min.
Rinse the membrane with Tris buffered saline (TBS) and place on the blot tray.
Acquire a blot image.

Blocking✔ StepInstructions

Actual Conditions

1

Immerse the membrane in 10 ml of EveryBlot Blocking Buffer.

2

Place on a rocker and block at room temperature (RT)for 5 min.

100
10
16,000
30

10 μl

25

100

300 V

350

5

3
4

High End of Range

1

Low End of Range

1

95ºC
5 min
10,000 x g
5 min

Low End of Range

3

Ideal Conditions

7 min

Ideal Conditions

5 min

High End of Range

10High End of Range

30

Watch our video —Quick Tips: How to Evaluate Western
Transfer Using Stain-Free Gels and a
Bio-Rad Imaging System

Mini-PROTEAN TGX Stain-Free andCriterion TGX Stain-Free Precast Gel Migration Guide
Mini-PROTEAN TGX Stain-Free Gels: 8.6 x 6.8 cm (W x L)
7.5%

Transfer

Immunodetection

11
22Electrophoresis and Western Blotting Layout

My Best Blot
Blotting Pro

EXPERIMENT
USER

33

DATE

Precision Plus Dual ColorTGS 30min
100v
MP TGX-SF
TYPE%4-15%BUFFER
TIME
VOLTAGEMW STANDARD
GELRemove the bottom sealing tape before you forget!
MEMBRANE TYPE

Trans-Blot PVDF 0.2µm

√

AMPS
VOLTS
TIME
7min
25v
2.5A
TYPE
VOLUME
TIME
5min
20mL
EveryBlot
1:10001hr
Antigen1
1º AB
1ST TARGETDILUTION
TIME
1:10001hr
Antigen2
1º AB
2ND TARGETDILUTION
TIME
1º AB
3RD TARGETDILUTION
TIME
1:10001hr
Antigen3√ √ √ √ √ √1º AB WASH TBSTBUFFER
VOLUMETIME
CYCLES
5min
10mL
2º AB
SPECIES AND
CONJUGATEDILUTION
TIME
GAM
HRP
1:10,000
1hr
StarBright
700
1hr
2º AB
SPECIES AND CONJUGATEDILUTION
TIME
1:10,000
1:10,000hFab
Rhodamine-Actin
1hr
2º AB
SPECIESAND CONJUGATEDILUTION
TIME√ √ √ √ √ √
5min
10mL
TBST
2º AB WASH
BUFFER
VOLUMETIME
CYCLESECL SUBSTRATE ClarityMax
TYPE
VOLUME
2mL

TRANSFER CONDITIONSBLOCKING BUFFER

NOTES

44
55
66
77
88

MW Standard

100
75

250
150
100

50
37

37

4–15%

37
25

250
150
100
75
50

25

37

20

25
20

20

15

15

10

15
10

50

100

250
150
100
75

75
50

4–20%

25

250
150
100
75
50

8–16%

Criterion TGX Stain-Free Gels: 13.3 x 8.7 cm (W x L)

Any kD

250
150
100
75

250
150
100
75

50

50

37

37

37
25
20
15
10

25
20

25
20

7.5%

15

15

10

10

250
150
100

10%

12%

250

250
150

150

100

4–15%

50

4–20%

250

75

150

150

75

50

100

100

50
37

37
50
25
20

37
25

Migration patterns (in kD) using Precision Plus Protein Unstained Protein Standards.

bio-rad.com/GetGels

250

100

75
75

8–16%

25

100

20

15

15

10

37
25
20
15
10

250
150
100

Any kD

75
50

37

37
25
25
20
15
10

250
150
100

75
50

75
50

37

25
20

20
15
15

10

10

Migration patterns (in kD) using Precision Plus Protein Unstained Protein Standards.

Available Well/Volume Formats
Mini-PROTEAN TGX Stain-Free Gel: 8+1 (30 µl), 10 (30 µl), 10 (50 µl), 12 (20 µl), 15 (15 µl), IPG (7 cm strip)■

Criterion TGX Stain-Free Gel: 12+2 (45 µl), 18 (30 µl), 26 (15 µl), prep+2 (700 µl), IPG/prep+1 well (11 cm strip, 550 μl)
Stain-Free imaging performed on ChemiDoc MP Imaging System. Activation time: 45 sec. Exposure time: 10 sec.

Request free magnetic guides forinterpreting protein gel migrationand protein standards.

■

BIO-RAD and MINI-PROTEAN are trademarks of Bio-Rad Laboratories, Inc. in certain jurisdictions. All trademarks used herein are the property of their respective owner.
TGX Stain-Free Precast Gels are covered by U.S. Patent Numbers 7,569,130 and 8,007,646.
21-0577 0921

15

Lysate 1

16

Lysate 1, K0

17

Lysate 218

Control

20

Lysate 2, treated

19

Blank

21

MW Standard

22

99

23

10

24

11

25

Request a free western blotting
layout sticky note pad.

26

12
13

12%

0
250
150

Bulletin 3255 Ver A

Electrophoresis

LANES

10%

0

Gel length, %Actual Conditions

1
2

Gel length, %Electrophoresis✔ Step Instructions

Visit bio-rad.com/GelLayout
for more information.

14
BIO-RAD is a trademark of Bio-Rad Laboratories, Inc. © 2022 Bio-Rad Laboratories, Inc.
Bulletin 3425 Ver A 22-0647 1022

BIO-RAD LABORATORIES, INC. | 15

Sample Prep

Electrophoresis

Transfer

Immunodetection

Image Acquisition

Image Analysis

References
Hammond M et al. (2013). A method for greater reliability in western
blot loading controls: Stain-Free total protein quantitation.
Bio-Rad Bulletin 6360.
McDonald K (2009). Overcoming the Coomassie blues.
Bio-Rad Bulletin 5939.
McDonald K et al. (2008). In-gel protein quantitation using theCriterion Stain-Free Gel Imaging System. Bio-Rad Bulletin 5782.
Taylor SC et al. (2013). A defined methodology for reliable
quantification of western blot data. Mol Biotechnol 55, 217–226.
Taylor SC and Posch A (2014). The design of a quantitative western
blot experiment. Biomed Res Int 2014, 361590.
Visit bio-rad.com/Stain-Free for more information.
BIO-RAD, MINI-PROTEAN, PRECISIONAB, and TRANS-BLOT are trademarks of
Bio-Rad Laboratories, Inc. in certain jurisdictions. All trademarks used herein are theproperty of their respective owner. © 2024 Bio-Rad Laboratories, Inc.
TGX Stain-Free Precast Gels are covered by U.S. Patent Numbers 7,569,130 and 8,007,646.

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