How to Quantify Western Blot Bands
A Western blot densitometry tutorial covering band selection, local background subtraction, loading-control normalization and Excel export.
Before you startUse original, lossless PNG or TIFF images with signals in the validated linear range. Check saturation in the acquisition software and confirm that the loading control is stable under your experimental conditions.
Illustrated guide
01Match target bands to the loading control
Open Strip intensity analysis from the Figure. Check row roles, target and reference assignments, and lane groups. Match each target lane to the same sample in the reference strip; verify membrane identity and lane order.
Use a same-membrane loading control for target/reference ratios. In phosphorylation mode, pair the phosphoprotein with the total form of that protein. Here, “total protein” means the corresponding protein band, not total-lane protein staining.
02Select band regions and subtract local background
Fit the blue signal regions around the complete bands without including adjacent lanes. Use a consistent region-selection strategy across the comparison. Place each green background region nearby, away from bands and without overlapping its signal region. Choose Save regions after manual adjustments.
How background correction works: For dark bands on a light background, corrected integrated density = N × B − ΣI. N is the number of signal pixels, B is the mean of the separate background region, and ΣI is the sum of signal-pixel intensities. For light bands on a dark background, the subtraction is reversed. See the measurement method.
03Normalize band intensity and export the results
Select Calculate ratios. Review target and reference background-corrected intensities, the target/reference ratio and any warnings before exporting Excel.
Normalized ratio = corrected target intensity / corrected reference intensity. If a denominator is invalid, review the reason for the missing ratio; do not replace it with zero.
Fold change is a further calculation: divide a sample’s normalized ratio by a defined control baseline. For arithmetic illustration only, a ratio of 0.60 divided by a control baseline of 0.30 gives 2-fold. Document the baseline and distinguish biological from technical replicates. A fold change is not a significance test.
Download the demonstration Excel workbook to inspect the result columns and review flags. Keep the project and original images with your analysis records.
After changing regions or reference assignments, review and calculate again. Saving regions preserves them when switching strips; it is not a per-strip approval requirement. A ratio cannot recover information lost to saturation or automatically remove differences between blots.
Method reference: Taylor et al. (2013), A Defined Methodology for Reliable Quantification of Western Blot Data.
Video guide
0:27This page uses simulated examples. The demonstration was recorded in build 2.1.3.