Ion exchange chromatography (IEX), pH, pKa and isoelectric point (pI) - cookie monster style! скачать в хорошем качестве

Ion exchange chromatography (IEX), pH, pKa and isoelectric point (pI) - cookie monster style!

Keep your ION the prize - pure protein! Lost? Use the isoelectric point (pI) to guide you and your protein of interest on your Ion EXchange chromatography (IEX) journey! IEX is a common protein purification technique in which we separate proteins based on charge. Proteins are made up of amino acid letters, some of which are sometimes charged (depending on pH). Different proteins have different amino acid spellings and thus different charges and we can exploit these different charges to separate them using columns filled with charged resin (basically little beads). ⠀ blog form with full text & figures: (refreshed from last July): http://bit.ly/ionexchangechromatography ⠀ ⠀ Unlike affinity chromatography (Ni-NTA, streptactin, etc.), which recognizes a specific feature of a specific protein (often a tag you genetically-engineer onto the protein when you express it) and thus is only useful if the protein you’re trying to purify has that feature, IEX can be used for “any” protein without relying on a tag. Therefore, you can use it on never-tagged proteins or proteins that were tagged but then you cleaved the tag off after the affinity chromatography step and now you want to purify the tag (and other lingering contaminants) away. Here’s how it works. First an overview and then more details on protein charge and practical advice on IEX. It’s a technique in which you separate proteins by their charge by getting them to bind to oppositely-charged resin (little beads) in a column, washing the other stuff off, and then, once all that stuff’s far away, pushing your protein off with salt. Since a salt (such as table salt, NaCl) is made up of a positive part (e.g. Na⁺ in table salt) and a negative part (e.g. Cl⁻ in table salt) that come apart when you dissolve them (it both dissolves and dissociates), it provides a source of both positively-charged & negatively-charged competitors. The more opposite the protein & bead charges are, the stronger the charge-opposite charge interactions, and thus the more salt it’ll take to get pushed off. So if you gradually increase the salt you can push proteins off based on their charge, separating them in the process. ⠀ ⠀ Because of that whole “positive part AND negative part,” a salt can be used to unstick either a negatively-charged (anionic) OR a positively-charged (cationic) protein (e.g. the Na⁺ can compete off, thus exchanging for, a positive protein and the Cl⁻ can compete off a negative protein). But in order for the protein to stick to the resin in the first place, that resin needs to be oppositely-charged compared to the overall protein. So if your protein’s +-charged (we call this cationic) you’ll want - charged resin (like an S column) but if your protein’s -charged (we call this anionic) you’ll want + charged resin (like a Q column).⠀ ⠀ I always had a problem remembering the terminology, but when it comes to ion exchange chromatography - the flavors refer to the type of ion that’s being exchanged - NOT the one that the resin - so anion exchange chromatography uses positively-charged resin to bind negatively-charged (anionic) proteins - and cation exchange chromatography uses negatively-charged resin to bind positively-charged (cationic) proteins. ⠀ ⠀ How do you know what to use? ⠀ ⠀ Whether, in which direction, and to what extent, a protein is charged depends on “how it’s spelled” (what protein letters (amino acids) does it have in what order?) and what the pH is. And the 2 are directly related - pH is a measure of how many protons (H⁺) are in a solution - it’s an inverse log, so the more H⁺ (more acidic), the lower the pH. Some amino acids are able to give protons (act as acids) or take protons, (act as bases) depending on the proton availability, and since those protons are positively-charged, give/take-ing them changes the protein’s charge. Gaining a proton (protonation) can positivize a neutral or neutralize a negative whereas losing a proton can negativize a neutral or neutralize a positive.⠀ For more practical protein-purification posts (and background/theory), check out the new page on my blog where I’ve collected some of my protein purification posts. http://bit.ly/proteinpurificationtech ⠀    more about Ago: https://bit.ly/agostructurestuff       more about all sorts of things: #365DaysOfScience All (with topics listed) 👉 http://bit.ly/2OllAB0 or search blog: http://thebumblingbiochemist.com