Western Blot Densitometry Analyzer
Lane densitometry with rolling-ball background subtraction and loading-control normalisation against GAPDH, β-actin or tubulin.
How it works
Lane intensity profiles are integrated after rolling-ball background subtraction, and target bands are normalised to a loading control from the same lane and the same membrane, with a user-chosen control lane fixed at 1.0 so every other lane reads as a fold change against it. Every pair of groups is then compared with Welch's t-test (or, where one side is a single control value, a one-sample t-test against it), and a Holm correction is applied across those comparisons by default. The per-lane table is corrected the same way. A band less than about 10% above background is not detected at all rather than reported as a small number, a detected band recovers about 9-10% low (a fixed cost of the integration window, measured on bands of known density), and a saturated band is flagged rather than silently quantified, since a detector that has stopped responding compresses real differences into a falsely small number.
Frequently asked questions
How do you quantify a Western blot correctly?
Integrate the area under each band’s intensity profile rather than measuring peak height, subtract local background from the same lane, and divide the target signal by a loading control measured in that same lane. Only compare normalised ratios between lanes, and confirm the signal is within the detector’s linear range — saturated bands compress real differences and make large changes look small.
Which loading control should I use for Western blotting?
Choose a control whose abundance is unaffected by your treatment and whose molecular weight is well separated from your target. GAPDH (~37 kDa), β-actin (~42 kDa) and α-tubulin (~50 kDa) are common, but all three change under some conditions — β-actin with cytoskeletal manipulation, GAPDH with hypoxia and metabolic stress. For treatments that may affect them, total protein stains such as Ponceau S are a safer normaliser.
Does this tool run statistics on my blot quantification?
Yes — once you designate a control lane, every other group is compared against it with Welch's t-test, and a Holm correction is applied across the comparisons by default when there is more than one, with SD/SEM/95% CI reported alongside the fold change. It will not quantify a saturated band or a lane assigned to two groups at once — both are flagged rather than silently included in the statistics.