Band Normalization

Normalization is useful for using one channel to correct for loading variation between lanes.

  • Normalization Channel: The Normalization Channel chosen for an image can be displayed in the Images table, Western Bands Table, or Western Lanes Table. Click Columns above and to the right of the Table section and select Normalization Channel in the dialog that opens.
  • Normalization Factor: Add the Normalization Factor to the Western Bands Table or Western Lanes Table by clicking Columns above and to the right of the Table section and selecting Normalization Factor in the dialog that opens.
  • Normalized Signal: Add the Normalized Signal to the Western Bands Table by clicking Columns above and to the right of the Western Bands Table and selecting Normalized Signal in the dialog that opens.

If the lane has a single band in the Normalization Channel, the Normalization Factor is calculated by dividing the signal for this single band by the maximum signal of all singleton bands in the Normalization Channel. Otherwise, the value is NaN. The value will be identical for all channels within a lane. The Normalized Signal column in the Bands Table is calculated as the Signal divided by the Normalization Factor.

This example will normalize the 800 channel to the 700 channel:

  1. Ensure only one band is present in each sample lane for the normalization channel.
  2. In the Normalize group, select 700 channel from the Normalize Channel list.

  3. Choose a background method in the Define Type list Define Type icon. See the Image Studio Software Help for more information on which background method to choose.

    Changing the background method will change the quantification values. When analyzing repetitions of the same assay, choose one background method to use each time repetitions of the same assay are analyzed.

  4. You should see the following in the Normalized Signal column of the Western Bands Table:
    1. Rows corresponding to bands in the marker lane will display "NaN" (because the marker lane has more than one band).
    2. Rows corresponding to bands in the normalization channel will display the value of the band with the greatest Signal value.
    3. Rows corresponding to bands in the opposite channel will show each band's Signal divided by the Normalization Factor from the band in the normalization channel in the same lane.