Having not touched them since … June maybe? Perhaps even May … I’m back to doing Western Blots, a technique that I wasn’t amazingly confident with to begin with. I did a dry run with samples that didn’t matter on Friday, knowing that Iw as likely to make tonnes of little mistakes all over the show, forgetting things that once seemed too intuitive to write down. What I need, I thought to myself, is an Idiot’s Guide.
There’s something that bothers me about Idiots’ Guides: They never tell you where something will go wrong. Or what will happen if it does. So here I present the Baking Biologist’s guide to Western blotting: The things that can go wrong, why they will go wrong, and whether to carry on or just scrap the whole thing.
1. Assemble the gel tank.
I’m lucky enough to work in a lab that uses pre-cast gels (because we’re really not a protein lab, and we don’t use nearly enough to cast our own). Take the gel out of the fridge and cut it out of its little plastic bag. (I have never yet been able to do this without thinking of draining mozzarella). Give it a good rinse with ddH2O and make sure you take off the white strip at the base. (Hang onto this, because when your gel won’t run you’ll start wondering Did I take it off? and while it’s possible to check and save your gel, it’s pretty tricksy.) Pop the gel(s) in the tank and add the bits of plastic that hold everything tightly together. The wells of the gel should be facing ‘inwards’. (So if you have two gels then the writing should be on the outside with the gels ‘facing’ one another. If you have one gel then when the tank is facing you the wells should be facing away from you). The buffer without antioxidant goes on the outside, and the buffer with antioxidant goes in the gap between the two gels. Both of them should be full to the top.
Potential horrible errors:
i) You put the gels in the wrong way around
I’m genuinely not sure how you’d do this, or how you’d not notice if you did, but presumably you’d figure it out the moment you tried to load the thing. In which case, take them out and start again. That will of course mean…
ii) You mix the external and internal buffers
Scale of disaster: Tiny. I’ve had to do this before to figure out why a gel isn’t running, and I didn’t notice any difference to the outcome. I think the only reason you actually prepare them separately is to save on antioxidant.
2. Prepare your samples and run the gel.
Mix sample with loading buffer. Heat to denature, say 85C for 5 minutes? (For some reason I always get a tonne of stuff coagulating on the bottom of the Eppendorf at this stage, so I add pretty much double the loading buffer I want in there to make up the volume). Remove the comb from your gel and pipette 20 uL or so into each well. Put the lid on, turn the voltmeter on, up it to 200V and off you go.
Potential horrible errors:
iii) You denature your protein for too long / not long enough / not at all.
Scale of disaster: Small. A bit of plasticity in denaturation time doesn’t seem to have effected my gels. I wouldn’t leave them there for 10 minutes, but I have left them for 7; and, equally, got bored after 3 minutes and just taken them out. Don’t have a melt down over this one.
iv) You forget to take the comb out.
Scale of disaster: Hilariously large until you figure it out. The second PAGE gel I ever ran was one weekend on my own with nobody else in the lab to ask. I spent an hour trying to figure out why my samples would not go into the gel. I thought maybe I was pipetting wrong. I thought maybe I needed to flush the gel through first (I was taught to do this when I learned how to cast gels a long time ago). I had to look up a YouTube video before finally the penny dropped: I had left the comb in. (In my defence, with an agarose gel I put the comb in and then I take it out. I don’t put a comb in a PAGE gel so it still perplexes me to find one there!)
v) Your gel does not appear to be running. The tank is making a strange ‘clicking’ sound.
Scale of disaster: Minimal. If the tank isn’t holding together properly then the buffer level can drop by flowing into the other chamber. If this happens then the current drops and the gel will stop running. Take the lid off, add more buffer, weigh the lid back down with something heavy (I find the block from a heat block pretty good) and start it off again.
vi) The bands are running in a curved shape (like a smile)
Scale of disaster: Minor. For the record, this is because you haven’t pipetted anything into your ‘blank’ wells. Your gel won’t be so pretty, but the results will be the same.