Analyzing Band Images from Separate Target Scans

Use the Separate Target Scan (STS) workflow when you need to analyze bands on blot images that were acquired in independent scans. Experiments involving a total protein stain should be analyzed using one of the total protein stain Experiment workflows.

This page contains general information about separate target scans. Read Quantitative Western Blot: Separate Target Scans Experiment for step-by-step information about the STS workflow.

Bands from independent scans cannot always be analyzed and compared accurately. This page provides guidelines, but researchers must decide how to analyze and compare bands in their experiments.

What Is a Separate Target Scan?

When a blot probed with multiple conjugated secondary antibodies (multiplex blot) is imaged, the signal for all detection channels is conventionally acquired during the same scan. You select all the channels to be imaged in the software, then click "scan" once. The imager then images the blot in a way that leads to an image being acquired for each detection channel. The process of allowing the image acquisition software and imager to acquire an image for each detection channel is called Conventional Multiplex Scanning.

On the other hand, a Separate Target Scan means repeating the imaging process for each detection channel to acquire images for each detection channel independently. You choose a detection channel in the software and click "scan", choose another detection channel and click "scan" again, and repeat the process for each detection channel.

Can My Images Be Analyzed in the Separate Target Scan Workflow?

To use the Separate Target Scan workflow, A) your internal loading control image must have been acquired in an independent scan from the target images, and B) you must be confident that bands from the independent images can be analyzed and compared accurately.

Part A: How Can I Tell if My Images Were Acquired Independently?

If you do not remember how a set of images was acquired, you can compare the image IDs to determine if the images were acquired together or independently.

• Images acquired by the conventional multiplex scanning method will have the same image ID.

• Images acquired in separate scans will have different image IDs.

The Internal Loading Control image ID can be different than the image ID for the protein target images, but all protein target images must have the same image ID. The image ID is the seven numbers that appear in the Image ID column in the Image Gallery. If the Image ID column is not in the Image Gallery, add it by clicking at the top right corner of the Image Gallery table.

Part B: Can Bands from My Independently Acquired Images Be Accurately Compared?

The researcher must choose whether to acquire images for multiple detection channels in the conventional way or separately. Using a separate target scan should most likely be avoided in the following situations.

After stripping and reprobing

After the blot has been stored for a long period of time

Conventional Multiplex Scan versus Separate Scan

Generally, it is best to use the Conventional Multiplex Imaging method to acquire images for all detection labels in the same scan. The checks in the following columns show why.