Western Blot Quantification
Measure band intensities from your blot image in a few clear steps: draw a box around each band row, check the lanes, name your groups — get normalised fold changes with saturation and linearity checks.
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How to use it
1. Open the original image from your imager or scanner — ideally the 16-bit TIFF, not a screenshot or an adjusted JPEG. 2. Draw a box around the target band row across all lanes, including some empty membrane above and below the bands. Then select the loading-control row and draw its box. 3. Set the number of lanes; use “Snap lanes to the bands” if lanes are unevenly spaced. 4. Name lanes and groups, untick ladders or damaged lanes and choose the reference group. 5. Read and export the per-lane and per-group tables, the chart, the annotated image and a methods paragraph.
What it computes
Signal is the distance of each pixel from the background colour (for dark bands, maximum value minus pixel value). For each lane, the tool builds the vertical intensity profile across the central part of the lane and integrates the band area above a straight baseline joining the background on either side of the band (the standard gel-analysis approach), or — as an alternative — subtracts the median of the box edges. Target values are divided by the loading control of the same lane and scaled so the reference group averages 1.0. Groups are summarised as mean ± SD and compared with Welch’s t-test. Pixels at the detector limit are counted in every lane, and lanes with a known protein load can be used to check that the signal is in the linear range.
Limits
Densitometry is semi-quantitative: it is valid only within the linear range of the detection system, so avoid saturated or overexposed bands and check linearity with a dilution series. Values are relative within one image; do not compare raw densities between blots or exposures. Housekeeping proteins can change with treatment — total-protein staining is often a better loading control. Lanes on one gel loaded from the same lysate are technical repeats, not biological replicates.
Keep your work
Nothing is uploaded or stored. Download the CSV, annotated image and settings file before closing the page.
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