If you’ve worked with SDS-PAGE and Western blotting for a while you’ll undoubtedly have seen bendy or fuzzy bands, so unlike those perfect razor-sharp rectangles shown on your antibody datasheet. While unattractive in their own right, these bendy bands make determining the molecular weight difficult and can interfere with band densitometry analysis, especially when using automated programs or when working with proteins that produce tight duplexes.
Fear not though, as there are typically five extremely simple steps you can take which will cure the vast majority of band bending, and make your blots presentable again.
1. Clean your kit
You’ve spent weeks growing your rare cells, treated them with your incredibly expensive novel compound, carefully harvested and lysed your sample and then cast your gel in between two dirty smeared plates. Ensuring that your plates and combs are spotlessly clean before casting is a great step to ensuring your perfect gels and perfect blots.
2. Shaken, stirred...
…and left to rest! A major issue with gel casting is improper mixing of reagents, in particular the acrylamide. In order to ensure a smooth separation of proteins across your resolving gel you want to ensure a constant acrylamide percentage throughout. But equally you want to get your gels cast and on with your experiment as quickly as possible. Here, as with many things in life, more haste equals less speed. A thorough mixing of gel reagents, followed by the equally important degassing step, will give you pristine gels first time, every time.
3. Fresh is best
In the same way, it’s worth keeping on top of your buffers and ensuring they’re fresh and importantly have the correct pH. We often have breaks in our Western blotting, and whilst it’s tempting to reach for that 6 month old bottle of running buffer, which has been sitting in the sun, it probably makes more sense to throw it out and start again. At the very least checking the pH of your stacking and resolving gel reagents is a must as this plays a fundamental role in how your proteins migrate through the gel.
4. Slow and steady
We know you want to get your exciting results ASAP, but running your gels with a high voltage might leave you having to cast and re-run your samples again. Forcing your samples through the stack too quickly can mean they hit the resolving gel spread out, rather than in a nice tight line. The same is true in the resolving gel, by running your gels at too high a voltage the buffers and your gels become hot, and this is a major contributor to warping and bending in your gel.
5. More isn’t always better
Less about bending and more about blotting, this step is vital in ensuring you get the most accurate results possible from your blotting. While it’s tempting to load the maximum concentration of protein in the maximal volume, sometimes it’s worth reducing the load to ensure you get a nice sharp band rather than an unintelligible blot. With new antibodies, and even new antibody batches, it’s always worth running a test with a standard curve of total protein. This simple step can save you sample, and time as it makes your analysis quicker and easier.
Following these five steps you help keep your bands razor sharp, making them the pride of your lab-book rather than something to leave tucked away in the back of your drawer.
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