What is the difference between Dot Blot and Western Blot?

Dot Blot skips electrophoresis — samples are spotted directly onto membrane. It is faster (2-4h vs 8-24h) and higher throughput, but cannot confirm molecular weight. Western Blot provides size verification via SDS-PAGE.

Research workbench

🔬Dot Blot Analyzer

Free online dot blot grayscale analyzer. Auto-detect membrane spots, measure intensity, background correction. No upload, runs in browser. Replaces ImageJ.

Drop image here or click to upload

PNGJPG

PNG, JPG · Max 10MB · Runs 100% in browser

What Is Dot Blot Analysis?

A dot blot (or slot blot) is a simplified immunoblotting technique where protein or nucleic acid samples are applied directly onto a membrane as circular spots, without electrophoretic separation. Quantifying the grayscale intensity of each dot allows researchers to compare relative amounts of target molecules across samples.

Traditionally, this analysis requires desktop software like ImageJ or commercial packages. This tool brings the same capability to your browser — upload your membrane image, auto-detect spots, adjust regions of interest (ROIs), and get publication-ready quantification results instantly.

How Grayscale Quantification Works

Each pixel in a grayscale image has an intensity value from 0 (black) to 255 (white). For a typical dot blot (dark spots on a light membrane), more protein means a darker spot, which means a lower pixel intensity. The tool calculates:

Mean Intensity — Average pixel value within the circular ROI.

Integrated Density — Mean intensity multiplied by the number of pixels (area). Accounts for both intensity and spot size.

Background — Median pixel value in an annular ring around each dot. Using the median makes it robust against neighboring dots.

Corrected Intensity — Background minus mean (for dark-on-light) or mean minus background (for light-on-dark). This removes membrane background variation.

Relative Intensity — Each dot's corrected value as a percentage of the maximum, for easy comparison.

Tips for Best Results

Use even illumination when photographing your membrane. Uneven lighting creates background gradients that affect quantification.

Avoid overexposure. If the tool shows a ⚠️ saturation warning, your image has pixels at maximum intensity (255), meaning the true signal may be higher than measured.

Include standards of known concentration on each membrane. Double-click a dot to mark it as a standard for absolute quantification.

Adjust sensitivity if auto-detection misses faint dots or picks up background noise.

Frequently Asked Questions

Is my data stored or sent to a server?

No. All image processing and analysis runs entirely in your browser using JavaScript and Web Workers. Your images never leave your device.

How accurate is this compared to ImageJ?

The quantification algorithms (mean intensity, integrated density, annular background subtraction) produce results within 5% of ImageJ's ROI analysis on the same image. The key advantage is convenience — no installation required.

What image formats are supported?

PNG and JPEG. Images larger than 4096×4096 pixels are automatically downscaled to fit within that boundary while preserving aspect ratio.

Can I analyze chemiluminescent (light-on-dark) images?

Yes. The tool auto-detects whether your dots are dark-on-light or light-on-dark. You can also manually toggle the polarity using the Dark/Light button in the toolbar.