Little things mean a lot
If you’re like me, you might sometimes wonder what would happen if you were to omit particular steps in an experimental protocol. I’m the sort who religiously follows instructions, even when I suspect they’re not that important, so I don’t often have the chance to find out.
Not so our poor rotating graduate student. This afternoon, he discovered exactly what transpires when you don’t wet your membrane in methanol prior to equilibrating in buffer and performing a Western blot.
Utter carnage.
Don't try this at home, kids
awww…. poor student.
I never wet my membranes in methanol, but I think I might use different membrane for my WBs 😉 Sometimes it is a valuable lessons, these “missed steps” – seldom cheap though.
I sympathize. The first time I ever used this new-fangled PVDF stuff, I didn’t know you *had* to wet it with methanol.
(Of course, once I figured it out I never went back to nitrocellulose).
PS “rotating graduate student” still makes me laugh.
The student was a really good sport, saying it was a learning experience and he was happy just to run it again. I think he’s got the right mental attitude for this job!
You have to take these things on the chin. At least you know why it went wrong. It’s when there are so many steps in a protocol and you have no idea which one you/someone else made a complete horlix of that it gets frustrating. At those points you just have to metaphorically sweep it under the carpet and pretend it never happened (don’t actually sweep it under the carpet because a) that wouldn’t be an appropriate health and safety approved disposal method and b) you’d actually have to go and find somewhere with a carpet), whilst hoping that it doesn’t happen again because then you really will have to try and work out why, and you know that is going to be a long and mostly profitless enterprise.
That middle one looks a bit like a colourful cormorant in flight, craning its neck downward.
I’ve butchered dozens of blots, especially during my PhD (actually, that’s where that monkey story came from), all the way from gel-making to film development. It’s actually one of the few “regular” lab techniques that have consistently avoided automisation. That’s why I think it still captures the essence of research: Tenacious perseverance, aiming for improvement in the face of bottled-up frustration.
That’s why, when blots work, they’re totally sweet.
Jenny,
Have you ever seen a student who didn’t, at one point, set the electrodes backwards and run the proteins off the gel into the buffer?! If it wasn’t a critical experiment, you can congratulate your student on having learned the lesson “on the cheap”, because I’m sure she/he will be especially careful in the future!
Steve
I have done this.
What’s a “Western Blot”? Is that something to do with “proteins”?