Western blotting is a tried and true way to detect and evaluate protein expression and is widely used by researchers. While it has been around for decades, Western blots are still presented as data in both scientific talks and in published manuscripts. Getting a Western Blot that is ready for publication is the goal but as many scientists can attest, it is not always easy. The 3 tips in this blog post will help you prepare your images for publication.
There are a number of steps in the Western blot process and many of these contribute to getting Western Blot images that are publication-worthy. For instance, if you do not have adequate blocking, then you can end up with lots of background noise. While you may essentially see the answer you have been looking for, if the image of the Western blot is not ideal, it can bring into question the validity of the result all together.
3 Tips for Publication-worthy Blots
1. Thoroughly Plan Your Experiment
We went into this in more detail in this article, but planning is key to having optimal Western blot images. Steps like validating the antibodies being used and ensuring all materials are not out of date can help with the overall success of the experiment. A big part of planning that affects publication is the order of your samples. Make sure you are loading the samples in a way that would be ideal for publication. For example, if you have extra lanes, you may consider adding in some additional samples to simply test if they too have a protein you will be probing for, but these samples have nothing to do with the experiment at hand. This can be a wise use of resources, but if done incorrectly, can jeopardize the chance of your Western Blot being used in a publication.
If you choose to add extra samples, add them at the end of the gel preferable with an empty lane separating them from the other samples. This allows you to easily remove them from the image for publication. If they are in the middle, then you will be forced to digitally cut them from the image, and it can raise suspicion when you have clearly pieced together the Western blot image. To avoid this altogether, be mindful of the order of samples.
2. Get a Variety of Exposures
A key part of getting publication-worthy Western blot images is the exposure and how the image is acquired. As a graduate student, I heard countless times about how I did not get the correct exposure of the Western blot. It took me a good while to figure out that it is really important to get a variety of exposures from faint to over-exposed. Having a number of exposures not only allows you to fully assess the data, but it also gives you a number of options for use in a publication.
If you’re using film, this is important because the way it looks on film may not be conveyed once you scan it so you want to have options to choose the one that best represents your data. This is one of the biggest pros of using an imager, like the Azure Sapphire. If you’re using an imager, then the imager will inherently capture a number of exposures and images for you to use from and this ultimately saves you a lot of time.
Instead of having to stand in the dark room doing a variety of exposures by hand at various lengths of time and hoping you chose the right ones, you can capture it with an imager in a matter of minutes and have a number of exposure options to choose from for your publication.
App note: Why You Should Leave the Darkroom
3. Label and Organize
This may seem obvious, but not all scientists are naturally organized and orderly, so it warrants being said. Being organized and having all things labeled is really important. Even if you do not think the experiment is usable for a publication or a talk, you never know if it will be needed later. With this in mind, always make sure to take the time to label each lane (right on the film if you did not use an imager), add the date, and make it very clear which experiment the exposures go with. You will likely perform many Western blot experiments. It can take years to get the data needed for a paper. Instead of trying to figure out which Western blot images go with which experiment you did 2 years ago, save yourself the time and headache by making sure everything is organized and labeled well the first time.
Another tip when creating figures is to make sure you denote which Western blot image was used in the figure, including the data and exposure chosen. There are many times when you may put together a figure for lab meeting or a poster at a conference, but even if it is not for a publication, make sure to note which experiment it came from. You may end up using this figure in the manuscript after all. This will save you time and stress in the end to have these details already determined.
With these 3 tips, you will set yourself up to get publication-worthy Western blot images every time. Incorporating these 3 tips into your Western Blot routine will prepare you for success when it comes to time to publish your data. What are your favorite tips? Share them with us!
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