Western blot densitometry

Guide to western blot densitometry

  1. Capture a linear image. Avoid over-exposure or crushed blacks. Use the saturation overlay below to check clipped pixels before quantifying.
  2. Upload your blot (JPEG, PNG, TIFF, or Bio-Rad .scn from Image Lab) or load the example. Colour images are converted to greyscale automatically. If the blot is tilted, use Draw straighten line along a horizontal reference, then Rotate to horizontal.
  3. Set background. Choose Global and draw one box on an empty lane/area, or Local to use a 2 px ring around each band.
  4. Draw band boxes tightly around each band, then use Select / edit box to move, resize, or rename ROIs. Use the same box height across lanes when possible.
  5. Read Adj IOD. This is the recommended value: background-subtracted total signal (responds to band darkness and area). Adj mean is intensity per pixel only — it does not reflect band size.
  6. Normalize for biology. Export CSV and divide target Adj IOD by a loading control (e.g. tubulin) in Excel or GraphPad. This tool does not replace biological normalization.

All processing runs in your browser; images are not uploaded to a server. For SDS lysis and blocking buffer recipes, see the western blot buffers guide.

Workspace

Background

Upload an image or load the example blot to begin
Band ROI Global background

Draw band box — drag on the image to add a ROI. Select / edit box — click a box to select it, drag inside to move, drag edges or corners to resize, then rename below. Draw straighten line — drag along a lane edge or marker that should be horizontal, then click Rotate to horizontal. Use the hand (pan) tool beside zoom to drag the view when zoomed in. Delete removes the selected box.

Results

Adj IOD (recommended) = background-subtracted total signal in the box — increases with band darkness and with ROI area. Adj mean = Adj IOD ÷ area (darkness only). Use the same box size across lanes when you want fair comparison of totals.

Name Area (px) Adj IOD Adj mean BG mean Raw IOD Mean signal

Draw band boxes to populate the table.