Background Subtraction for Blots and Gels

This page provides a quick look at background subtraction options for blot and gel analyses. You can refer to the background subtraction guide for complete information.

Background Options for Analyzing Blots and Gels
(available without additional keys)

Method

Best Use Cases

Method Comparison

Background Calculation
Average or Median (Local Methods)

  • If only a few shapes need to be quantified, adding the shapes individually and using local background methods can work well.

  • Choose Median instead of Average to reduce the impact of outlier pixel intensities in the background.

  • If shapes are in a lane containing many nonspecific bands and the background is uniform around the shapes, local methods may produce better results than the Image Studio Lane method.

  • The Average or Median background method computes the average or median intensity of pixels in a border around a shape and subtracts that background from the shape. The width and position of the border can be adjusted.
User-Defined
  • Background must be fairly uniform throughout an image.
  • Use when Image Studio Lane is not available and closely-spaced shapes will make it difficult to use local methods without having background border overlap shapes.
  • Use when the background is uniform throughout the image and bands are so close the background border from local methods might overlap a band.
  • Background is often not uniform enough for User-Defined to accurately represent background for every shape in an image.
  • The User-Defined background method finds the average pixel intensity within a shape or shapes placed by the user to calculate background. When multiple shapes are used, the average pixel intensity in each will be averaged together to find the background.

 

Background Options for Analyzing Blots and Gels
(requires Western Analysis Key)

Method

Best Use Cases

Method Comparison

Calculation
Lane

  • When Western, MPX™ Western, or DNA Gel applications are in use

  • If there are many shapes and the shapes are close together

  • In lanes with smearing between bands

  • Could be better than local methods in cases where background shapes from local methods might overlap other shapes or there is smearing in a lane

  • Background is calculated by drawing a linear connection between areas of minimum brightness between shapes in a lane and subtracting the noise beneath these lines

Example

Recommended Method(s)

Other Method(s)

Median

Local background methods are appropriate, since only a few, widely spaced shapes need to be quantified. Use Median to reduce the impact of spurious bright pixels around the shapes (see arrows).

Lane

The Image Studio Lane method will work, if available.

Average

Average may be skewed by outlier bright pixels surrounding the shapes.

User-Defined

The highly variable background means User-Defined is not the best choice.

Median or Average

The background does not contain spurious bright pixels, so Average or Median may be used. Be careful that the background border does not overlap a nearby shape.

Lane

The Image Studio Lane method will work, if available.

User-Defined

The uniform background means User-Defined may be employed. However, local methods are preferable, because they sample background directly surrounding shapes.

User-Defined

Closely-spaced shapes might make it difficult to keep the background border from local background methods from overlapping an adjacent shape. The uniform background means the User-Defined method can be a solution.

Lane

The Image Studio Lane method will work, if available.

Average or Median

The background border from local methods could overlap another shape.

Lane

The Image Studio Lane method is ideal for subtracting background from many close together shapes in a lane.

Average or Median

The background border from local methods could overlap another shape.

User-Defined

User-Defined might not accurately represent background caused by smearing in the lane.