Western Blots: The Good, The Bad and The Ugly…

…by Sophie / from the United Kingdom / studies PhD Tissue Repair / 4th Year

Striding into the lab with a cowboy swagger, the scientist plants her feet and stares down the opponent. Tucking back her lab coat, she spins her pipette deftly in its low slung holster. She knows there can only be one winner in this fight. Her adversary returns the steely glare… it is the western blot apparatus.

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 “Howdy pardner” Meet The Western Blot

Looking at the beautiful neat bands in many papers you would perhaps assume that the western blot is a simple, fool-proof technique. You would be wrong. They are the snake in your boot.

After recent repeated skirmishes with them, I’m here to share my own failures in the hope of preventing yours. Though not the most challenging in terms of skills needed, the western blot has taken down scientists of greater ability than me, so let’s not be lone rangers in this – circle the wagons and share our hardships.

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“There’s not enough room for the both of us in this gel” Planning your western blot.

Swing open the saloon doors and take the challenge head on. Have your ammunition ready for the fight: What proteins do you need antibodies to? How much will you need to dilute your samples? What volume will each of your gel wells hold… no really how much? The box might say 50µL but like the sheriff who tells you he’s taken down ten bandits at once, ask around to see what the real deal is.

“Load the wagons” Running your gel.

Head to the bar and select your poison (buffer). There probably won’t be a bartender with a Southern drawl to make it for you but I don’t know your lab so maybe there is. Set up your gel in your bucket full of buffer and pipette in your samples.

Just as a cowboy wouldn’t leave a barrel of his gun unfilled, nor should you leave a gel lane unloaded. The proteins wont run down the gel evenly and the ‘smile’ it leaves will not be on your face I can assure you.

Plug in your voltage – you might find it good to get things going with a low voltage then move to a higher one. Check for bubbles so you know there’s actually some electricity flowing and head off to get some grits, whatever they are.

“Stick ‘em up!” Getting the proteins onto the membrane.

If your loading buffer has hit the bottom of the gel then you’re ready for round two. If it hasn’t, well keep firing ‘til it does.

Slosh out the trough and refill with a whole new buffer, layer up a sandwich with the gel and a membrane as the delicious sciencey filling and absorbent papers and furry scotch pads as the bread. It is ridiculously essential to put the sandwich the right way round in the bucket so the proteins transfer in the right direction. This is literally the one mistake I have yet to make because I am so paranoid about it, but bandits can storm the convoy at any time so I have to remain vigilant.

“3…2…1… Draw!” Remove your membrane.

That dreaded moment when you peel the membrane from the gel and reveal whether the proteins have transferred successfully is pretty nerve-wracking no matter how sure you are it was set up right. If it’s on the absorbent whatman paper and not on the membrane, accept defeat and know you live to fight another day.

If it’s on the membrane, yee-haw! Giddy up partner. Wash it in yet another buffer then in a blocking solution.

“Wanted… dead or alive” specific antibody targeting.

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Snip that membrane up so you can stain for the different proteins you’re interested in. Hopefully you’ve got a validated antibody but frankly that’s a whole other fight in a whole other town. Dilute the primary antibody in blocking solution and immerse your membrane in it. Strap the nosebag on your horse and head home. Next day, wash away and then add your secondary antibody… I’ve ended up with completely null results after using the wrong secondary but it’s not like poisoning the water hole, you can always wash it off and try again.

“You looking at me?” Developing your western blot.

This part is the killer shot. The two days of splashing about in various buffers may have been completely in vain if you mess something up here. No pressure.

I mean what could go wrong now? Take your membrane, add your substrate, saddle up your steed and dash to the dark room or the scanner depending on your chosen method of punishment.

Just… do not forget what went where. Do not forget to line up your sheet with the ladder and draw it on. Do not for goodness sake accidentally mix up your western developing substrate kit reagents A and B with your protein assay kit reagents A and B, pipette the wrong thing on and mess up the entire membrane…

“You’re my favourite deputy”

I truly hope your westerns are peaceful, you lasso the data you hoped for and you can return to the ranch triumphant.

 

 

This blog post was originally published on 23/08/17 here.


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