2023 Scientific Publication Requirements for Western Blots and Gels

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Western Blotting

You’ve worked hard on your research, so when it’s time to submit your work for publication, don’t forget to check the publication requirements for Western blots for each journal. With over 3,000 publications worldwide featuring our products, you could say our users know a thing or two about publishing. The imagers and systems from Azure Biosystems deliver the quality journals are asking for, but image capture is only the beginning. When capturing images of gels and Western blots, it is imperative to record relevant information to ensure an image meets the requirements for journal publication.

Autoradiograph of an SDS-PAGE gel using phosphorimaging from Azure Sapphire
Figure 1. The above figure was published in the Journal of Cell Biology and shows an analysis of phosphorylation levels on wild-type and mutant Dam1 complexes. (A) Autoradiograph of an SDS-PAGE gel (8–14%). (B) Coomassie blue–stained SDS-PAGE gel (8–14%). Licensed under CC BY 4.0.

General publication requirements for Western blots and gels

Unfortunately, publication requirements for Western blots (Figure 1) and gels are not always uniform. Some journals recommend formats other journals will not accept, so always check the guidelines of the specific journal you plan to submit before preparing your draft for review! Knowing this, you can plan your experiments so you don’t need to cut apart the image of your gel or blot and reassemble it to create a figure. Avoid cutting and cropping your images more than necessary. Make sure internal controls are included on every gel or blot. Multiplex fluorescent Western blots are an excellent way to image your control with one or more targets on the same blot.

Some journals accept a variety of image types in the submission stage, but impose strict formatting rules once the manuscript is accepted. At the submission stage, some journals simply require figures be legible and provided in a format that is compatible with a broad range of operating systems and visualization programs. Others recommend figures be in the preferred publication format throughout all stages of the submission process.

Once a paper is accepted for publication, the requirements for Western blots and gels become much more specific and vary from journal to journal. In general the following guidelines will help ensure your image is publication ready:1

  • Capture images with at least 300 dpi and at least 190 mm wide.

    You can always shrink an image if it is too large, but you cannot increase the size of an image and many journals explicitly ban "upsampling." Using the Azure 600 Western blot imager makes capturing and saving large files easy.

  • Save a raw version of the image with no manipulations, including brightness and contrast.

  • Keep a record of the settings used to capture the image (resolution, exposure time, etc.)..

  • For modified versions of the image, keep a record of all manipulations that were performed to achieve that version of the image (brightness adjustments, channels overlaid, etc.).

Two scientists working on Azure 600
The Azure 600 uses a 9.1MP camera to provide high resolution imaging perfect for publications. Change the sample to optics distance using adjustable height shelf for enhanced detection. Zoom into the area of interest with ROI imaging to reduce background.

Journal-specific publication requirements for Western blots and gels:

You should be aware of specific requirements for Western blots and gels when capturing images and preparing figures. Each journal will provide general guidelines for file types, file size, resolution, and color mode which apply to all images, including gels and Western blots. A list of general guidelines for publishing Western blots and gels in popular journals, such as ElsevierNatureScience, and Wiley are outlined below:

Best practices for preparing gel and Western blot images for publication

Creating figures from images of gels and Western blots (Figure 2) often presents a challenge. Authors must balance presenting the most relevant lanes and regions of a gel or blot with providing an accurate representation of the “big picture” of the experiment. Careful attention in the experimental design phase can help simplify this process. When planning the layout of the gel, think about how the data will be presented in an eventual figure and arrange samples in a logical manner. Do not include extraneous or unrelated samples in between the samples you plan to compare.

Western blots captured using Azure c600, published in nature
Figure 2. This figure was published in nature and shows Western blots captured using Azure c600 from Pascini et al. Immunoblotting evaluating the tissue-specific expression of PAI-1 in the transgenic mosquito.

Whenever possible, comparisons should only be made between samples run on the same gel. Internal controls, housekeeping protein standards, or total protein stains should always be processed on the same gel as the experimental samples. Therefore, in the planning phase, consider ways to optimize the amount of data to be generated from running just one gel. If one gel is not possible, make sure to include a control on each gel.

A recent publication by Kroon et al examined published Western blot figures and found a majority are cropped, missing essential information about the methods, and do not supply the original images as supplementary information. This reading provides recommendations to make Western blot figures more informative and reproducible, such as minimizing cropping and including (and labeling) molecular weight markers in all images.

Two scientists smiling holding Azure pub mugs
Are you getting ready to submit your paper for publication? Congratulations! We want to recognize your hard work. If you have published using an Azure Imager, post your publication on social media using the hashtag #ImagedbyAzure and tag us, we'll send you a pub mug to show off!

Editors at the Journal of Cell Biology published an article in 2004 addressing the temptation to alter or “beautify” images and describing acceptable and unacceptable manipulations of digital images. It provided an overview of the guidelines for blot and gel images that had been published to date by a variety of journals. Adjustments applied evenly across the entire image such as adjustments to brightness, contrast, or color balance are generally acceptable; however, it is always preferable to use an image that does not require such adjustments. For example, if your bands of interest in your Western blot are faint, it is better to take a longer exposure for publication rather than choose to digitally adjust the faint image to increase perceived band strength.

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Nonlinear adjustments to an image should be avoided if you’re submitting to a journal. If they are used, carefully document the adjustments that were made. Some journals will require that these adjustments be described in the methods or figure legends. It is never acceptable to digitally alter the data in an image of a gel or blot; do not adjust contrast to hide background or faint bands. Those “nonspecific” bands may indicate your Western blotting conditions were not ideal and you need to change your blocking buffer or adjust your antibody concentrations. Alternatively, such bands maybe contain data whose importance will only become clear in the future. Maybe that “extra” band is actually an isoform of your protein, or a cleavage product.

Always save the original images used to make a figure. Some journals will request original images during the review process. Some journals, like the Nature portfolio journals in the life sciences, require original, unprocessed images of gels and Western blots used in figures be published as Supplementary Information (Figure 3).

Journals vary in how they prefer to receive figures in initial submissions. Always look up the specific requirements for the journal to which you are submitting, including required naming conventions for figure and image files. In summary, remember to capture high-resolution images (at least 300 dpi) and to carefully record your imaging settings. If you adjust an image, keep track of exactly what changes you made and always maintain a copy of the original raw image.

If you’re looking for an imager that’s reliable and provides sharp, clear images with every scan, look no further than a system from Azure Biosystems. Browse this list of available systems and choose the system that works best for your studies, like the Azure Sapphire Biomolecular Imager. The Sapphire is a next generation NIR fluorescent scanner equipped with lasers that delivers unmatched flexibility and performance for phosphor imaging, Western blots, animal imaging, in-cell Westerns, and more. Learn more about the Sapphire by clicking here.

SOURCE
  1. Rattan, U.K.; Kumar, S.; Kumari, R.; Bharti, M.; Hallan, V. Homeobox 27, a Homeodomain Transcription Factor, Confers Tolerances to CMV by Associating with Cucumber Mosaic Virus 2b Protein. Pathogens 2022, 11, 788. https://doi.org/10.3390/ pathogens11070788