Rafiki’s Western

You have to have suffered the torment of Western blotting to appreciate this cartoon and it’s nigh on impossible to really explain the job to someone who hasn’t. Although please feel free to have a go below.

Western blotting is integral to almost any experiment that involves proteins whether it’s validating an antibody, the success of a knockdown or identifying binding partners. And yet it all hinges on the fickle nature of having a decent antibody one of the most misunderstood and abused areas of modern research. Entire mythologies evolve in labs about the exact set of circumstances required not just to get bands but at the right size too. There are descriptions of Elven lore in the Lord of the Rings that are less esoteric. And then of course there’s the desperate rationalisation of why the bands are at the wrong size. Hmmm, probably glycosylation was always my personal favourite.

It doesn’t matter how hard you stare…

There are two common outcomes which show up scientists who ought to know better. One is the horrible smiley, smeary blot, usually presented by a PhD student with a thousand yard stare that hints at long nights and weekends spent optimising every step of the protocol to no avail. Either their supervisor doesn’t have the heart to tell them to give it up or is a sadist. Either way, the blot is meaningless. The second is the blot that has been cropped so tight that it is literally just a band and becomes totally meaningless. Annotating it with a size marker doesn’t make it OK!

The situation is not helped by the opacity of antibody suppliers as to which application a given antibody works in. Catalogues are full of ‘representative’ blots that exhibit the sins above and yet still we part with our hard-earned research funds in the hope that this time things will be different. There is also the in-house antibody made by the lab publishing the suspiciously neat data. Sure they’ll send you an aliquot…eventually. What do you mean it doesn’t work? It’s fine in our hands. Are you sure your Tris was pH 7.245?

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