SDSPAGE an overview

>> YOUR LINK HERE: ___ http://youtube.com/watch?v=xP7vJnTSNrs

SDS-PAGE stands for Sodium DodecylSulfate PolyAcrylamide Gel Electrophoresis. It’s a technique for separating proteins by size (length not shape as we’ll see). If you take a biochemistry course, it’s probably one of the first experiments you do. But it can become so “routine” that it’s easy to forget and/or take for granted the awesome science going on at the molecular level. Which is really sad because the science is super non-sad. It’s really really cool! So, put on your biochemistry goggles cuz it’s time to think like a molecule! • P.S. Today I got to teach my first student to run his first SDS-PAGE gel! Something I’ve always been so excited about! • text old, video new • blog form: http://bit.ly/sdspageruler • Sodium DodecylSulfate PolyAcrylamide Gel Electrophoresis is one of the most frequently used techniques in biochemistry. We use it to separate proteins by their lengths by using electricity to send them swimming through a gel mesh. • Longer proteins get tangled up more and travel more slowly, so when you stop their swimming they won’t have traveled as far and when you stain the gel to show their location they’ll be higher up the vertical gel slab. For this protein character counter to work, your “input file” (protein) has to be in the proper format (unwound and negative-charged), so let’s take a look at how we do it! • Proteins have different lengths, shapes and charges, so if you want to separate based on length alone you need to erase the shape (denature them) and “paint over” the natural charges with an even negative charge (you don’t want to just erase the charge cuz you need a negative charge to motivate them to move through the gel towards the positive end). • Just like all poems are written using letters from the same alphabet but they can be dramatically different, proteins are all made up of letters (amino acids) from the same protein alphabet but, depending on what combinations of letters are there, they can have wildly different properties and are thus best suited for different purposes. Would you read the Iliad or the Odyssey to a group of preschoolers? Or Cat in the Hat to college students (I hope so on this one :P). • Also like poetry, • proteins • can take • different shapes • What if you want to know how long a poem is (how many letters it has)? As we saw above, you can’t just “count the lines” (shape size can have different relationships). Instead you need to get all the poems in a single long line format (e.g. proteinscantakedifferentshapes). This requires unfolding the protein, removing its “natural shape” and hence “denaturing it” • A key thing to note is that all of these letters (even if they look like they’re on “different lines”) are strung together LikeCursiveLetters through strong peptide bonds in their generic backbones. But different letters also have unique “side chains” (aka “R groups”) sticking off with different properties (small, big, +, -, etc.). We looked at each of these through #20DaysOfAminoAcids http://bit.ly/allaminoacids • And speaking of charge… we’re going to use the “opposites attract” strategy to motivate proteins to move through the gel. But proteins can have different charges – certain letters can be + or – charged and different proteins have different numbers of those letters (e.g. imagine that the dots on i’s are + charges the – on t’s are – charges. since different poems have different numbers of them, they’ll have different overall charges). And this can “confuse” our character count apparatus. • LENGTH is what determines how fast they move ( thus how far they’ll have traveled when you stop the experiment) but CHARGE is the motivating force that makes them even try to move in the 1st place. So you need to make sure that charge size are directly related • So we need to remove the shape, keep it removed, and “standardize” the charge so that all the proteins are – charged (and thus motivated to move towards the + bottom of the gel) and that charge is directly related to the length of the protein not to the identity of the letters. So what kind of “erasers” and “charge paint” do we need? • Let’s start with the shape erasers - The shape comes from different letters interacting with each other (e.g. those negative “crossed t’s” wanting to hang near the positive “dotted i’s”) and with the liquid (often water) they’re bathed in (e.g. water-loving (hydrophilic) letters wanting to be on the surface water-avoiding (hydrophobic) residues wanting to stay hidden in the core). But the more energy the letters have, the more likely they are to explore! And heat is a form of energy, so we can raise the temperature to denature the proteins. • finished in comments

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