Blot naming: Southern, western, northern, etc.
In elementary school I learned “Never Eat Soggy Waffles” to remember the cardinal directions - North, East, South, West - I still think it in my head when I’m trying to orient myself. But no one gave me a guide for blotting directions - you might have heard of the western blot. It’s a technique that’s used a lot to detect specific proteins. But did you know there’s also a Southern blot (which came first), a northern blot, and eastern blot, and some more copy-cats?! So today I present to you the bumbling biochemist’s “compass” for biochemical blots! blog form (text old, video & some graphics new) : http://bit.ly/blotcompass No matter what “direction” is in the name - the basic premise is the same - take a mix of biomolecules and separate (typically by size) then transfer to a membrane and probe to analyze. If the probe finds a match, a band you will see, but what kind of molecule will it be? For western blots, you’re looking for specific proteins, Southern blots look for specific DNA, and northern blots look for specific RNA. And they utilize different types of “probes” to do so - antibodies for western blots, and little pieces of sequence-complementing DNA for the others. The western blot is a way to test for the presence of specific proteins and it’s named after the Southern blot which tests for specific pieces of DNA - and the “Southern” name comes from the name of the scientist who invented the technique, Edwin Southern. This is why you capitalize the S but not the w. The western blot isn’t the only technique to hop onto the direction name train - there’s a 3rd main type of blot called the northern blot and, which uses DNA to probe for RNA. Edwin Southern invented his blot in the 1970s because he wanted to be able to isolate the genes responsible for making 5S ribosomal RNA (rRNA) - most enzymes are proteins, but the ribosome (protein-making machinery) is a mix of proteins & RNAs, with the RNAs doing the brunt of the work.And Southern wanted to find the genes for these RNAs to give them their due credit! (and study how they work…) Since DNA is usually present as big ole chromosomes you start by chopping it into smaller pieces using restriction endonuclease (DNA scissors that recognize & cut at specific sequences), then run it through a gel. At the time, the traditional way to go about it would be to extract all those cut up DNA pieces out of the gel (basically cut out the bands and melt away the gel around them). And then bind each of those purified bands to a filter and probe each of the filters with complementary DNA probes. Southern thought - the probing part’s good, but why not just transfer all the bands to a filter at once? As long as you preserved their relative locations you wouldn’t lose any information - you’d lose less DNA since you didn’t have to go about purifying it first (where you’d inevitable lose some yield at each step) - and you’d save a lot of time. Southern gives credit for the “blotting” part of the name to Frederick Sanger - the name might sound familiar because we looked at him a couple times - in addition to sequencing the first protein (insulin in 1955) he developed the “Sanger Sequencing” techniques for sequencing DNA using chain-terminating nucleotides - these dideoxynucleotides can get added but not added onto because they lack a 3’ OH and by adding some terminators of just a single letter you can puzzle out the sequence. http://bit.ly/2koF5el Sanger used electrophoresis to separate RNA fragments on cellulose acetate strips, then transferred the fragments out of the strips and onto DEAE paper by “blotting through” It’s actually kinda funny because a bunch of different scientists wanted to join the directional naming fun - but there are only 4 cardinal directions - so you ended up with a ton of methods being called “eastern blotting” which turns out to not be so funny because it can instead be confusing… As well as as some other “hybrid” directions. Here’s a sampling… Most of the techniques the eastern blot name refer to are variants of the western blot that look for post-translational modifications - “bells and whistles” or “cherries on top” added to the proteins after they’re made (translated) - things like phosphorylation (addition of negatively-charged phosphate groups), glycosylation (addition of sugar chains), or lipidation (addition of lipids (fatty things)) far-eastern blot: lipid analysis - this one’s a bit more different - instead of gel electrophoresis it uses TLC (thin layer chromatography) which basically uses capillary action to “suck” liquid through a special sheet that different components in the liquid interact with differently so they travel at different rates and thus separate as they go - kinda like when a piece of paper with a marker mark gets wet and the colors in the marker dye start to separate. After the TLC, the lipids are blotted onto a PDVF membrane Finished in comments
