Antibody Validation Experiment Steps

Antibody Validation is critical for Qualitative Western Blot Assays and Quantitative Western Blot Assays. See and Quantitative Western Blot Overview for more information.

Name

To help with record-keeping, give every Experiment a distinct name. The description field provides space for you to include important details about the Experiment.

Set up images

Once the correct images have been chosen, enter information for the target antibodies.

Click the zoom buttons above the image to make the displayed image smaller or larger . A zoom level, shown as a percentage relative to the size of the software window, is shown next to the zoom buttons.

Define lanes

The Define lanes page identifies lanes on the image for analysis. To find lanes, draw a boundary that encloses all the lanes on the image.

By default, Auto mode automatically performs lane finding. Manual mode is also available.

Tips for Drawing the Boundary

Draw the boundary around all the lanes, even if you do not plan to use all the lanes for your analysis. If necessary, you can exclude lanes from the analysis later in the workflow (for example, on the Add lane details page).

Examples

Correct Placement

Incorrect Placement

Comparison of Lane Finding Methods

Automatic Lane Finding

By default, Empiria Studio uses a patent-pending process called Adaptive Lane Finding (ALF) to automatically find the number of lanes and the precise boundaries of each lane.

Figure 212. When the Auto button is bright blue, Auto mode is active and lanes will be found automatically using the Adaptive Lane Finding process.

For more information on the Adaptive Lane Finding process, see the Calculations Section.

Manual Lane Finding

Lanes can still be found manually (if necessary), providing consistency with previous versions of Empiria Studio. In Manual mode, you choose the number of lanes and place lane lines manually.

For more information about the differences between automatic lane finding and manual lane finding, see the Calculations Section.

Add lane details

  • Name: Provide a lane name to facilitate record keeping and organization.

  • Type: Specify the appropriate type for each lane.

    • MW Marker: Choose MW Marker for all lanes that contain a molecular weight marker.

      Designate marker lanes now to size bands at a later step in the analysis.

    • Positive: Choose Positive for lanes that contain a positive control.

    • Negative: Choose Negative for lanes that contain a negative control.

    • Sample: Choose Sample for lanes that you want to use but do not want to identify as positive or negative.

    • Not Used: Choose Not Used for lanes you do not want to include in your analysis. For example, you might choose Not Used if a lane is empty. Empiria Studio will not find bands in lanes marked Not Used.

  • Value Label: Indicate the amount, concentration, dose, or treatment. Also indicate the associated unit.

    Click the Autofill button to see options for quickly filling out the table.

Assign MW marker

Assign a molecular weight marker to estimate the molecular weight of bands. Estimated molecular weights will be available in the Analysis Table of the Quantify bands page.

You can estimate the molecular weight of target bands in one channel using a marker in another channel when you link a Band Channel to a Marker Channel.

Assign Marker

To assign a marker, choose a marker from the list and add band boxes to at least three marker bands.

Use Existing Marker from the MW Marker List

  1. Select a marker from the list.

    Markers available from LI‑COR are available in the list by default.

  2. Click Add to add band boxes to bands in the marker lane.

    Once three band boxes have been, the channel will be checked in the Channel list. This check indicates that the marker is ready to estimate molecular weights in subsequent analysis steps.

Add New Marker

You can add other markers to the list, and markers you add will be available in other Experiments. To add a new marker:

  1. Click New.

  2. Enter a name for the marker.

  3. Enter the first molecular weight and press Enter.

  4. Continue adding molecular weights until all weights have been added, then click Create.

    The molecular weight marker will be saved and assigned to the existing image for band sizing.

After a marker has been created, it cannot be edited. If you entered the molecular weight marker incorrectly, add a new marker with the correct information.

To size bands in one channel using a marker in different channel, link the channel that has the bands to be sized (the Band Channel) with the channel that has the marker (the Marker Channel).

The 700 channel is used as the marker channel for both the 700 and 800 channels.
Figure 213. With these settings, a marker in the 700 channel will be used to size bands in the 700 channel and 800 channel.

Delete a Marker

You can delete markers that were added to the list, but you cannot delete markers that come with Empiria Studio.

  • The deleted marker will be removed from the list in the current Experiment.

  • The deleted marker will not be available in Experiments you create after deleting the marker.

  • The deleted marker, and band size data based on that marker, will be saved in other Experiments where the marker has already been used.

To delete a marker:

  1. In the MW Marker section, click Information.

  2. In the MW Marker Information dialog, click Delete.

  3. Click Yes to confirm that you want to delete the marker.

Quantify bands

The Next button will not be active until you have identified a band in each lane (aside from lanes that you marked as Not Used or MW Marker on the Add lane details page).

Find and Quantify Bands

The Find Bands button will identify and quantity a band in every lane, except in lanes that you marked as MW Marker or Not Used on the Add lane details page.

  1. Ensure the correct channel and band box are displayed for your analysis.

    • Click the Channel Display button above the image to change which channels are displayed.

    • Click the Band Display button above the image to change which band boxes are displayed.

  2. Select a protein name from the Protein list.

  3. Click Find Bands.

  4. Position your mouse over the image.

    A horizontal line will appear.

  5. Locate the horizontal line as close as possible to the bands of interest, then click.

    Band boxes will be placed around bands in lanes near the horizontal line.

Add Individual Band Boxes

Click Add to identify individual bands one at a time. Bands cannot be added to lanes marked as Not Used or MW Marker.

  1. Ensure the correct channel and band shapes are displayed for your analysis.

    • Click the Channel Display button above the image to change which channels are displayed.

    • Click the Band Display button above the image to change which band boxes are displayed.

  2. Select a protein name from the Protein list.

  3. Click Add, then click on the band.

    A band box will be placed around the band.

  4. Continue clicking bands until you have added all the band boxes you need to add, then click Done.

Manually Adjusting a Band Box

  1. Before adjusting a band box, ensure your image display is set so that you can clearly see the entire band.

    Click Image Display above the image to adjust the image display. Ensure some background is visible on the image so that you can completely see the edges of the band.

    Avoid manually adjusting a band shape until you have carefully examined the band under appropriate image display settings.

  2. Click the band box to select it.

  3. Adjust the border of the band box by dragging anchor points, or click and drag inside the selected band box to reposition it.

Delete Band

Click the band box you want to remove and click Delete.

View Data

To view quantification data, click the Analysis Table.

  • Click Export above a table to export the data in the table in CSV or Excel (XLSX) format.

  • Customize which columns are included in the table by clicking in the table and selecting the columns you want to include.

  • Click a column header to sort the data in the table by the values in that column.

  • Click and drag column headers to rearrange the order of columns in the table.

Review and report

The Review and report page allows you to export a PDF file containing the data and image from the validation or export the image and data table individually. The data in the report is organized into sections. Click to expand a section to view data and export options.

Images

  • Click above an image to export the image. Images can be exported as a high-resolution TIFF for publication in a journal or as a PNG appropriate for slide presentations. Do not use these images for analysis.

  • The units for image size can be set to English or metric on the General Options page. See General for more information.

  • Click Hide features to hide lane and band rectangles. If lane and band rectangles are hidden, they will not be included in the exported image or report.

Table

Click Export above a table to export the data in the table in CSV or Excel (XLSX) format.

Export Menu Options

On the right side of the window, there is a menu with three export options.

  • Click PDF Report to export a PDF of the images and data on the Review and report page.

  • Click Experiment File to export all the images, quantification steps, and data from this Experiment to a single file that can be imported into Empiria Studio on another computer.

  • Click All Report Images to export all images from the Experiment to separate files for use in print publications or digital media (such as a slide presentation). Do not use these images for analysis.

    In the export dialog, click Browse to find a parent folder for the images and enter a name for a new folder in the Folder Name field. The images will be exported into the new folder, which will be inside the parent folder.

Rate Experiment

Rate the Experiment for your records. This is an opportunity to record if the Experiment went "well" or not, depending on the specific requirements for your research.

Figure 214. Your rating will appear in the Experiment list for the Project.

All Antibody Validations are stored in the Antibody Validation Library. If you open an Antibody Validation from the Experiment List in a Project, you see the Review and report page. Click Edit in Library to view the other steps in the validation or to modify the validation.

Done

Click Done to mark this Experiment as complete and return to the Experiment List.