WESTERN BLOTTING

Western Blot Normalization
Using Image Lab™ Software
Quick Start Guide

Total Protein Normalization Using Stain-Free Gels
This guide describes the steps to normalize your chemiluminescent blot with stain-free technology.

1Create a multichannel image and normalize your western blot■■

With the stain-free and chemiluminescent blot images open, select Lane and Bands (Figure 1)■■

Select Add channel in the Normalization Channel dropdown menu (Figure 2)■■

Drag the stain-free blot to the Normalization Data channel and click OK (Figure 3)

Fig. 3. Create a multichannel image of the stain-free andchemiluminescent blot images.
Fig. 1. Normalization is foundunder Lane and Bands in theAnalysis Tool Box.

Fig. 2. Add
normalization channel.

■■

■■

Select the appropriate band detection settings based on blot band intensity (Figure 4).
Image Lab Software will automatically detect lanes and bands
If needed adjust the lane frame by using the Adjust Frame and Resize Frame buttons (Figure 5). Adjust theindividual lanes by selecting the lane that needs adjustment and moving the anchor points (Figure 5)

Note: When lanes are manually moved or adjusted, previously detected bands are removed for any lanes that are adjusted. Goto the Bands tab, click Detect Bands..., then select the previous appropriate band detection settings to add these bands again.

Fig. 4. Automatic lane and band detection.

Fig. 5. Manual lane adjustment.

© 2017 Bio-Rad Laboratories, Inc.

■■

In the Normalization Lane dropdown menu, select a lane to use for normalization.
Do not use a lane with a protein standard as the normalization lane (Figure 6)

Note: The sample in the normalization lane is typically the null or wild-type condition in an experiment and is considered to
be 1x or onefold. All lanes will be compared to this null condition. True protein normalization will happen when comparing thechemiluminescence signal in each lane to its corresponding stain-free lane.

Fig. 6. Select normalization lane.

2

View analysis results■■

View the normalized volumes by selecting Analysis Table from the main toolbar (Figure 7). All calculations will beperformed by the software, including the normalization factor and normalized volumes. The chemiluminescent 		
blot channel intensity values are now adjusted for variation in the protein loading between different lanes. This willallow accurate comparisons of target protein intensities across all lanes of a gel■■

From the Analysis Table tools, click Display data options to customize the data table (Figure 8)■■

From the Analysis Table tools, click Export analysis table to Excel for additional analysis (Figure 9)

Fig. 8. Customize the data table.

Fig. 9. Export the data to Excel.

Fig. 7. Analysis table with the calculated
normalization factor and normalized volumes.

Bulletin 6434

Once exported to Excel, the results can be arranged as shown in Table 1■■

Table 1. Intensity values for protein identified in the chemiluminescent blot.
ChannelLane NumberBand Number

Volume (intensity)

Normalization Factor

Normalized Volume (intensity)

Chemi

Lane 2

13,860,064

13,860,064

Chemi

Lane 3

13,406,560

1.29

4,416,034

Chemi

Lane 4

12,331,168

1.89

4,408,112

Chemi

Lane 5

13,782,112

1.05

3,981,556

Chemi

Lane 6

13,383,328

1.314,445,670

Chemi

Lane 712,444,832

1.97

4,822,224

Chemi

Lane 813,445,536

1.09

3,739,923

Chemi

Lane 912,851,872

1.38

3,934,866

Chemi

Lane 101

1,940,544

2.03

3,937,656

Note: The Normalized Volume (intensity) value represents each chemiluminescent signal (band) that is properly total
protein normalized to the stain-free signal in its corresponding lane. This is the value you should use for further analysis and,ultimately, publication of experimental results.

Visit bio-rad.com/web/ImageLabNormalization for more information.

Excel is a trademark of Microsoft Corporation.

Bio-RadLaboratories, Inc.
Web site bio-rad.com USA 1 800 424 6723 Australia 61 2 9914 2800 Austria 43 1 877 89 01 177 Belgium 32 (0)3 710 53 00 Brazil 55 11 3065 7550
Canada 1 905 364 3435 China 86 21 6169 8500 Czech Republic 420 241 430 532 Denmark 45 44 52 10 00 Finland 358 09 804 22 00
France 33 01 47 95 69 65 Germany 49 89 31 884 0 Hong Kong 852 2789 3300 Hungary 36 1 459 6100 India 91 124 4029300 Israel 972 03 963 6050
Italy 39 02 216091 Japan 81 3 6361 7000 Korea 82 2 3473 4460 Mexico 52 555 488 7670 The Netherlands 31 (0)318 540 666 New Zealand 64 9 415 2280
Norway 47 23 38 41 30 Poland 48 22 331 99 99 Portugal 351 21 472 7700 Russia 7 495 721 14 04 Singapore 65 6415 3188 South Africa 27 (0) 861 246 723
Spain 34 91 590 5200 Sweden 46 08 555 12700 Switzerland 41 026 674 55 05 Taiwan 886 2 2578 7189 Thailand 66 2 651 8311
United Arab Emirates 971 4 8187300 United Kingdom 44 020 8328 2000

Life ScienceGroup

Bulletin 6434 Ver CUS/EG17-0710

0717

Sig 1216