By Our Mass Spectrometry Guru Jon Reed
This will be the first in an ongoing series of technical tips for those interested in biochemistry and mass spectrometry. Anyone involved with these fields knows there can be considerable headache and heartache that goes hand-in-hand with even the simplest of experiments. Along the way, I’ve picked up quite a few tricks that I feel should be shared with others. Most of these are pretty simple, and are intended to do one or more of the following: save time, save money, and improve your experiments. I’d like to keep these segments informal. After all, this is a blog and not a review article or method journal.
One recurring theme you will find will be (hopefully) the destruction of the “because we’ve always done it that way” mentality that pervades many laboratories. This way of thinking runs counter-intuitive to everything the progressive nature of science strives to achieve, and stems from laziness and ego- neither of which have any place in a well-functioning lab. Let’s face it though, it’s going to be hard to get rid of lab egos. We’re all nerds. Some nerds think they’re not nerds. Deal with it.
OK, now on to this week’s topic….
Western Blotting: Is methanol the be-all, end-all solvent for Western blotting buffers?
NO. Not by a long shot. It’s pricey and toxic. You can find cheaper substitutes on the shelves of Walmart and Sam’s Club (if you live in the U.S., that is) that work just as well, if not better than MeOH – and you’re not paying the high prices charged by chemical companies. It’s also ACS grade when sold in stores like that, so you don’t have to worry about it being junk.
So why is MeOH used almost exclusively for blots?
“BECAUSE WE’VE ALWAYS DONE IT THAT WAY!”
I’m certainly not the first person to think of this, but I figure it warrants repeating. Simply substitute isopropanol (IPA) for MeOH when preparing your Towbin’s buffer recipe. Remember that most stores sell it at a 70% or 90% concentration, so be sure to adjust your volumes! You can also use IPA to wet your PVDF membranes.
I’ll post some western blots run in IPA vs. MeOH, as well as images of the stained PVDF membranes so you can see the results for yourself. What’s the point in changing things if they’re not as good or better than the original, right?
Other solvent sins: No matter what solvent you’re using for westerns, at no point should you use HPLC-grade solvents for blotting. Good grief! They certainly won’t ruin your experiments, but they will definitely ruin your budget, and I can’t tell you how many times I’ve seen labs doing this as a matter of routine. Electrophoresis-grade buffers make perfect sense, but HPLC-grade solvents? They make perfect nonsense. Find a lesser-grade solvent in your catalog or simply head to the local megamart and compare prices (that’s right, you have to do some of the work here too).
So, if you’re running mass spectrometry experiments, now is not the time to be cheap with your solvents. If you’re western blotting, that’s a different story. Be bold, be cheap, be happy.
Next week: preparing frits for nano-LC columns.