Ion exchange chromatography protein purification and isoelectric point (pI)

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).

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.

  
  

[wabnilu]

Thank you for the very informative video. Your effort is much appreciated!!

[satonobre]

a really great and rare explanation!!! keep it up champ!

[GeorgeCronigen]

Really nice I got two questions as always. a)Why there is this trend of using an anion exchange column when our PI is relatively low and using a cation exchange column when our PI is relatively high? We could as well when we have a low PI for example 5.5 to create an acidic buffer making our protein overall positively charged and use a cation exchange column. But of course in this case we can protonate the resin too. So is this the reason of the specific trend? b) also I am a little bit confused with the preparation of the column. You say that we equilibriate the column with salt but that's just before we place our sample onto the column. So what is the reason for that? The only reason I can think of is that while we prepare our resin or even if the resin is prepared us there already molecules that bind to some degree to the negatively or positively charged beads, but we use again this thing after we have placed our sample onto the column again we add salty solution to make w/e weakly binding to the column molecule to elute. So if these are well said why don't we just skip the equilibriation before adding the sample and keeping just the wash that comes after the sample placement? Thanks again fro everything.

[MohammedAli-bjjk]

Just subscribed to your channel. I found it very informative. Thanks for the invested efforts