Western blotting quantification

Introduction

Western blot quantification (densitometry) turns band darkness into numbers you can plot. You have imaged the blot, it looks fine in the figure, and then the boss asks for quantification, numbers, a graph and an n=6 replicates! This page is The Bond Lab free western blot densitometry tool. Just draw boxes on your bands, subtract background, and read off the adjusted optical density (Adj IOD) in the browser. No ImageJ wrestling, no nightmares converting your jpg into a 16 bit TIFF, it runs locally on your machine, quickly and easily. We built it for the quick “is lane 3 actually stronger?” check.

WHAT IT IS FOR

A western tells you if a protein is there and, on a good day, whether one lane looks darker and bigger than another on the same membrane. Densitometry is just turning those grey bands into values you can plot on our graph. Think of it as aking how many pixels in the box are darker than the background and how much darker are they? We use this page when we are comparing treatment vs control on one scan, checking a phospho band against total or loading control, or sanity-checking a result before opening desktop software. It is also handy for showing students what background subtraction means on a real blot.

You need a flat image file a JPEG, PNG, TIFF, or a Bio-Rad .SCN file. It will not fix a badly designed experiment or a dodgy blot probed with a messy antibody from that supplier we all love to hate. You still want replicates, a sensible loading control and exposure that is not blown out. And it will not auto-divide your band of interest by your housekeeper protein. You can export the table and do that normalisation yourself in excel.

WHAT IT DOES

Upload your scan or image file (colour is fine, the tool will convert to greyscale) or hit Load example blot to poke around with a lovely blot we prepared earlier. If the image is wonky, draw a straighten line and Rotate to horizontal (this will clears your boxes, so do it first). Pick Global background and mark one empty bit of membrane, or Local and the tool will estimate background in a 2 px ring around each box. Draw band boxes, zoom in, rename them whatever you want and read the results in the table. Turn on the saturation overlay if you suspect the imager ate your highlights; the black/white sliders only change what you see on screen, not the maths. Export CSV or a JPG with the boxes drawn on. For lysis and blocking recipes upstream of all this, see our western blot buffers guide.

HOW IT WORKS

Each pixel becomes a grey value; we sum everything inside your box. That sum is raw IOD — bigger or darker bands score higher. Global mode: one background box, its average grey is subtracted from every band (as if that background filled the whole ROI). Local mode: a thin ring just outside each band, which helps when background creeps across the blot but lanes are cramped.

Adj IOD is the number we actually use. This is the total background-subtracted total signal in the box, so both how dark and how big the band is. Adj mean is the same correction per pixel only; it ignores area, so do not compare fat and skinny bands with mean alone. Keep box height similar across lanes if you want a fair fight. Saturation highlighting flags pixels stuck at pure black or white; if half the band is red or flagged black, rescan with gentler exposure and try again.

HOW TO USE IT

Start with a capture image of the blot that is not saturated. You use Highlight saturated pixels and trust your eyes. Upload, straighten if needed, set global or local background, then draw band boxes tight to the signal. Select / edit box lets you nudge and rename. Read Adj IOD in the results table, export CSV when happy, then in Excel or GraphPad divide your protein of interest by tubulin (or total stain) from the same blot. This tool does the rectangle maths; you still own the biology and the stats across separate experiments.