Western blot figure maker, with the uncropped originals

$$\begin{array}{rl}& \text{band traced from }(x_1,y_1)\text{ to }(x_2,y_2)\text{:}\\ & \theta =-\mathrm{atan}2(y_2-y_1,x_2-x_1)\end{array}$$$$\begin{array}{rl}& \text{strip pixel }(u,v)\text{ is read from}\\ & \text{the original at}\\ & \mathbf{p}=\mathbf{c}+R(-\theta )\left(\begin{array}{c}u-w/2\\ v-h/2\end{array}\right)\end{array}$$$$\text{shown}=255\times \frac{v-\text{black}}{\text{white}-\text{black}}$$$$\text{pixels}=\frac{\text{width (mm)}}{25.4}\times \mathrm{DPI}$$$$\text{ppi of a strip}=\frac{\text{crop width (px)}}{\text{width (mm)}/25.4}$$

Straightening. A blot photographed or scanned slightly turned has bands that slope. Trace one band (or the row of wells) from one side to the other: the line from $(x_1,y_1)$ to $(x_2,y_2)$ in the original image slopes by $\mathrm{atan}2(y_2-y_1,x_2-x_1)$, taken between −90° and 90°, and the image is turned by minus that angle, rounded to 0.01°, so the band runs level. You can also type the angle, use the slider or ±0.1° steps, or turn by 90°. Positive angles turn clockwise. Turning by anything other than a multiple of 90° resamples the pixels (bilinear interpolation), which slightly softens them; the original file is never changed.

Crops in original coordinates. You draw each crop on the straightened image, where it is an upright rectangle $w\times h$ pixels. The tool stores it in the pixels of the original file: the rectangle $x,y,w,h$ turned by the straightening angle about its centre $\mathbf{c}=(x+w/2,y+h/2)$. Every pixel of the strip is read from the original through that rotation (the formula above), so the strip and the outline on the uncropped original are the same region by construction, and the corners can be listed exactly. Crop each blot from the left edge of the first lane to the right edge of the last, the same on the target and the loading control, so that the lanes line up when the strips are stacked. Leave room above and below the band: JBC asks for molecular-weight markers above and below the band of interest, and in a survey of 551 papers most blots were cropped so tightly that the markers could not be seen (Kroon et al. 2022).

Contrast, linear only. Each raw value $v$ (8, 16 or 32 bit) is shown as $255\times (v-\text{black})/(\text{white}-\text{black})$, clipped at 0 and 255, and optionally inverted ($255-$ that) so bright chemiluminescence bands print dark on light. That is one straight line applied to every pixel of the image, the adjustment image-integrity guidelines accept when it is applied to the whole image and does not hide or create features (Rossner & Yamada 2004; Cromey 2010). There is no gamma, curve, sharpening or local edit. A 16-bit image starts at its own minimum and maximum, which clips nothing; an 8-bit image starts at 0–255, unchanged. For each strip the tool reports the share of pixels pushed past the black or white point (flattened) and the share at the file’s maximum value (saturated on the detector), because a saturated band can no longer be measured. Quantify on the raw values, not on the figure: see western blot quantification in ImageJ and free western blot quantification software, or the Blots tab of LabCanvas Image Analysis.

Stacking and lanes. All strips are scaled to the blot width you set (50 mm by default), keeping their shape, and stacked with a small gap. Lane centres are fractions of that width, evenly spaced to start with (centre of lane $i$ at $(i+\frac{1}{2})/n$); drag them onto the lanes if a gel ran unevenly. Each row above the lanes is one line of text: names or values separated by commas (horizontal, at 45° or vertical), a + and − row typed as −+++−+++ (one character per lane), or groups typed as siControl ×4 | siKinase ×4, which centres each name over its lanes with a line beneath. A row’s label (such as “EGF”) goes on the left. To label a single gel image rather than build a stacked figure, the gel and western blot lane labeller is quicker.

Molecular-weight markers. Choose the ladder you ran (PageRuler Plus, PageRuler, Precision Plus Protein Dual Color, or type its sizes) and tap its bands on each image, or type each band’s y position in pixels. The marks are kept in original-image pixels; any that fall inside a crop become a tick and size on the left of that strip, at the band’s real position, without interpolation. Labels that would overlap the one above are left out while their ticks stay. To read the size of an unknown band from the ladder, use the western blot molecular weight calculator.

Source data for the journal. The sheet of originals shows each image whole and unrotated, with every crop drawn as its turned rectangle in red and numbered as in the figure, the ladder marks, the exposure, the display mapping and the crop coordinates. The ZIP holds the figure (PNG and SVG), that sheet (PNG and SVG), the crop and exposure list as text, each original at full size with its crops outlined, and the original files themselves, byte for byte (the text records each file’s SHA-256, so an editor can check it is the same file). Journals ask for this in different words:

Image-integrity screening finds problems in published blots often enough to matter: in 20,621 papers from 40 journals, 3.8 % contained inappropriately duplicated images, many of them western blots (Bik et al. 2016). Showing exactly where each strip came from removes the question. See also what you may and may not do to microscopy and blot images.

Output. The PNG is drawn at the DPI you choose ($\text{pixels}=\text{mm}/25.4\times \mathrm{DPI}$) and records it in the file. The SVG is sized in millimetres, embeds each strip at its own pixel size as PNG, and writes every label, tick and line as text and vector lines in Arial, so they stay editable in the LabCanvas figure editor, Illustrator or Inkscape. If a strip has fewer than 300 pixels per inch at the chosen width, the tool says so: scaling up adds pixels but not detail.

Sources: Kroon C, Breuer L, Jones L, et al. Blind spots on western blots: assessment of common problems in western blot figures and methods reporting with recommendations to improve them. PLoS Biol 2022;20:e3001783, doi:10.1371/journal.pbio.3001783. Rossner M, Yamada KM. What’s in a picture? The temptation of image manipulation. J Cell Biol 2004;166:11–15, doi:10.1083/jcb.200406019. Cromey DW. Avoiding twisted pixels: ethical guidelines for the appropriate use and manipulation of scientific digital images. Sci Eng Ethics 2010;16:639–667, doi:10.1007/s11948-010-9201-y. Bik EM, Casadevall A, Fang FC. The prevalence of inappropriate image duplication in biomedical research publications. mBio 2016;7:e00809-16, doi:10.1128/mBio.00809-16. Pillai-Kastoori L, Schutz-Geschwender AR, Harford JA. A systematic approach to quantitative Western blot analysis. Anal Biochem 2020;593:113608, doi:10.1016/j.ab.2020.113608. Journal policies: the pages linked in the table.

The tool opens with this example: two 16-bit chemiluminescence images, 640 × 400 pixels each, with a PageRuler Plus ladder in lane 1 and eight samples (siControl and siKinase, each treated with EGF for 0, 5, 15 and 30 min). The p-ERK image sits turned 1.4° and the GAPDH image -0.8°.

1. Straighten. Tracing the p-ERK doublet from $(131.9,214.2)$ to $(569.8,224.9)$ gives a slope of $\mathrm{atan}2(10.7,437.9)=1.40°$, so the image turns -1.40°. The GAPDH band traced the same way gives $0.80°$.
2. Contrast. The p-ERK image’s values run from 1,041 to 45,684, so those are the black and white points and nothing is clipped. A pixel of 23,000 is shown at $255\times 21959/44643$ $=125$, or 130 once inverted so the bands print dark on light.
3. Crop. Each crop runs from the left edge of lane 2 to the right edge of lane 9 and from about 60 to 30 kDa. In original pixels:

Their corners in the original images (top-left, top-right, bottom-right, bottom-left of the strip): strip 1 (pERK-blot.tif) (101.0, 178.9), (601.8, 191.1), (600.0, 264.1), (99.2, 251.9); strip 2 (GAPDH-blot.tif) (100.5, 182.5), (601.5, 175.5), (602.5, 248.5), (101.5, 255.5). The rectangles lean against the image edges by the straightening angle, which is how they are drawn on the sheet of originals.

4. Labels. Three rows: siControl ×4 | siKinase ×4 as groups with a line under each, −+++−+++ labelled “EGF”, and 0, 5, 15, 30, 0, 5, 15, 30 labelled “Time (min)”. The 55 and 35 kDa ladder bands fall inside both crops, so each strip gets those two ticks; “p-ERK1/2” and “GAPDH” go on the right.
5. Size. At 50 mm wide, each 501-pixel strip has $501/(50/25.4)=255$ pixels per inch. The PNG at 600 dpi is $50/25.4\times 600=1,181$ pixels across the blot, so the strips are scaled up about 2.4×; the tool notes that the strips are below 300 ppi at this width. Text is 7 pt.
6. Export. Download the PNG for the manuscript, or send the SVG to the figure editor to add the quantification. Then download the source-data ZIP: the sheet shows both images whole, the p-ERK crop as a rectangle turned 1.40° against the image edges, numbered 1, and the GAPDH crop numbered 2.