Gel analysis - Creating boxes around curved lanes

Hi! I am analyzing a few vertical gels that have two bands fairly close to one another. I can get the boxes just fine, but they automatically adjust to be flush with the first box. Normally, that is ok, but in one of my gels, the samples are curved a bit. So, if I want to just box the top band, I can capture the first two or three, but the rest of the boxes start to pick up the second band. Is there a way to turn off the automatic positioning of the boxes or to set a path/line for them to follow or something?


Dear @engineer16,

welcome to the forum.
I am not sure how exactly you do the Gel analysis but since I have seen many mistakes happen in this task since most people trust in what a google search lists top most (e.g. putting small boxes around the band). You might want to have rather first a look on several publications regarding Western blots (as listed below) while many things will also be important to consider when measuring signals from a gel.
Furthermore if the gel or Western blot image is tilted or the band are not straight or show a bending, ImageJ is not the best option to analyze those (and I generally prefer to solve all image analysis tasks using ImageJ). Therefore, you might want to have a look at the GelAnalyzer2010 software which is also free and gives you the possibilities to analyze bands according to the best-practice standards as shown also in this video

[1] S. C. Taylor, T. Berkelman, G. Yadav, and M. Hammond, “A defined methodology for reliable quantification of Western blot data.,” Mol. Biotechnol., vol. 55, no. 3, pp. 217–26, Nov. 2013.
[2] S. C. Taylor and A. Posch, “The design of a quantitative western blot experiment.,” Biomed Res. Int., vol. 2014, p. 361590, 2014.
[3] J. E. Gilda and A. V Gomes, “Stain-Free total protein staining is a superior loading control to β-actin for Western blots.,” Anal. Biochem., vol. 440, no. 2, pp. 186–8, Sep. 2013.
[4] J. E. Gilda, R. Ghosh, J. X. Cheah, T. M. West, S. C. Bodine, and A. V Gomes, “Western Blotting Inaccuracies with Unverified Antibodies: Need for a Western Blotting Minimal Reporting Standard (WBMRS).,” PLoS One, vol. 10, no. 8, p. e0135392, 2015.
[5] M. Landry and A. V Gomes, “Antibodies: Half of samples fail protein-blot tests.,” Nature, vol. 529, no. 7584, p. 25, Jan. 2016.
[6] R. Ghosh, J. E. Gilda, and A. V Gomes, “The necessity of and strategies for improving confidence in the accuracy of western blots.,” Expert Rev. Proteomics, vol. 11, no. 5, pp. 549–60, Oct. 2014.
[7] J. E. Gilda and A. V Gomes, “Western blotting using in-gel protein labeling as a normalization control: stain-free technology.,” Methods Mol. Biol., vol. 1295, pp. 381–91, 2015.
[8] M. Gassmann, B. Grenacher, B. Rohde, and J. Vogel, “Quantifying Western blots: pitfalls of densitometry.,” Electrophoresis, vol. 30, no. 11, pp. 1845–55, Jun. 2009.
[9] F. Heidebrecht, a Heidebrecht, I. Schulz, S.-E. Behrens, and a Bader, “Improved semiquantitative Western blot technique with increased quantification range.,” J. Immunol. Methods, vol. 345, no. 1–2, pp. 40–8, Jun. 2009.
[10] J. Bordeaux et al., “Antibody validation,” Biotechniques, vol. 48, no. 3, pp. 197–209, 2010.
[11] A. Degasperi, M. R. Birtwistle, N. Volinsky, J. Rauch, W. Kolch, and B. N. Kholodenko, “Evaluating strategies to normalise biological replicates of western blot data,” PLoS One, vol. 9, no. 1, 2014.
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