If you’ve worked with SDS-PAGE and Western blotting for a while, you’ll undoubtedly have seen bendy bands, or fuzzy bands, or both! This blog post will help you achieve 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, here are five extremely simple steps you can take to cure the vast majority of band bending, and make your blots presentable again.
Get Rid of Bendy Bands SDS-PAGE
STEP 1: Clean your gel running system
Picture this: you’ve spent weeks growing your rare cells, treated them with your incredibly expensive novel compound, carefully harvested and lysed your sample, only to cast your gel in between two dirty smeared plates. Yikes! To save yourself from this woe, always clean your plates and combs.
STEP 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.
STEP 3: Fresh is best
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.
STEP 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.
STEP 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.
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Following these five steps will help you keep your bands razor sharp. They’ll be the pride of your lab-book, rather than something to leave tucked away in the back of your drawer. While you’re here, watch out for these assumptions about SDS-PAGE from our friends at NanoTemper.
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