Western blotting is used in a variety of research fields to used to detect how much of a protein is in a given sample. If you are new to Western blotting, then you are likely in the stage of learning all you can about how the procedure works and the specific steps to ensure you get accurate and valuable data. Or maybe you have been doing them for awhile, but your results are less than ideal. You might be finding yourself repeating experiments because of different issues keep coming up. In this blog post, we are going to talk about what to consider before you do your next Western blot, so you do not fall into the common issues that can arise due to poor planning and preparation.
Ready to plan your Western blot experiment? Here are 6 steps to follow before getting started:
STEP 1: Ensure you have everything you need- and nothing is expired
While this may seem obvious, it is definitely worth the reminder. As scientists, we can get rushed and sometimes that leads to trying to cut corners (especially true in those grad school and postdoc years). But as many have learned, cutting corners does not save time in the end and instead usually causes things to take longer.
Using a digital imager is another step to getting great results with your Western blots. Digital imaging provides a much larger dynamic range compared to film; low- and high-intensity bands can be imaged simultaneously. It produces a file that is immediately ready for publication, while sheets of exposed film must be photographed or scanned to generate a digital image.
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Take the extra time to make sure you have all of your materials, you have adequate amounts (check with other lab members to make sure someone else is not planning a big experiment around the same time), and your buffers are fresh, and not expired.
Skipping this step risks the sample, integrity of your data, as well your entire experiment; it is always smart to make sure your ducks are in a row. Take care of it on the front end to avoid questioning your results over something trivial, such as using an old buffer.
STEP 2: Choose the correct antibodies
While the antibody company has assured you their antibody works well for your protein when used in Western blotting, you will still want to validate the antibody yourself in your hands with your exact Western blot set up in the lab. This will give you the confidence in the results you see if you know the antibody is specific and produces little background. Or if not, then at least you know what to expect.
Another consideration when choosing primary antibodies is crossreactivity. Doing a simple test Western Blot ensures there is not any crossreactivity. When choosing secondary antibodies, consider the species and any cross reactivity that might be prevalent there.
STEP 3: Load the proper controls
It may be tempting to leave out a control to save more lanes for your samples, but choosing the necessary and proper controls for your experiment is always important. Skipping a control can require you to have to repeat the experiment again once reviewers come back with a request.
Think ahead to which controls will be needed in order to affirm the results are accurate and what a publication may require.
STEP 4: Choose the right gel percentage for your proteins of interest
Choosing the right gel percentage is especially important if you are looking for multiple proteins that are a similar size on the same membrane. This step ensures that the gel adequately separates the proteins to allow for reliable detection.
STEP 5: Plan the order of protein detection
You will likely be probing for at least 2 proteins: the protein of interest and a loading control. Most of the time, you will want to probe for multiple proteins of interest to make your sample go as far as possible. In both cases, plan which protein you will probe for first and then subsequent proteins and their order.
STEP 6: Create a sample order that makes sense for publication
Even if this is the first Western blot for this study, set it up as though it could be used in a publication because it very well may if you play your cards right. You don’t want to get to the end of the experiment to find you detected the proteins you were looking for and you have a great result, but the order of the lanes does not make sense for publication purposes. To give as many options as possible to choose from for publication, consider the order of the samples on the front end to save time.
Do I really need to spend time planning my Western blot experiment?
The short answer is: yes! As many graduate students and even post docs will think, they do not have the time to plan out the experiment. There are so many other experiments to get to and this is just one of many.
Even though it seems logical to just move forward and get the data you want, the extra time to thoroughly plan and design your experiment ahead of time will prevent countless hours of frustration trying to get the Western Blot data you are needing.
Here are some compelling reasons you want to put some time into planning and preparing your Western blot experiment:
If you’ve only got a limited amount of sample or your sample is precious (i.e. patient samples or cancer tissue from a mouse model that took months to develop), then you want to ensure the Western blot is done correctly and well the first time.
The truth is, we’re all busy. Planning out your experiment will save you time in the end. This could mean getting a publication out there sooner or not getting scooped. Plus, Western blots can take a long time from beginning to end. You want to set aside the time needed. Nothing is more frustrating than telling yourself you’ll be finished by 4 p.m. only to realize you don’t have enough buffer and now you have to take 30 minutes to make that.
There are a number of steps involved in Western blotting and any one of those can have issues. Planning will help you avoid the pitfalls so you do not get halfway through an experiment and have to throw it out to start all over again because you realized you forgot something.
As with every experiment, you want your data to be accurate. Planning ahead of time can help you make sure your data is both accurate and publication ready.
Using the 6 steps above, you will be able to design your next Western blot experiment with the best chance of avoiding common pitfalls. Taking time to plan with these things in mind will go a long way in saving you time, money, and frustration.
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