Ion exchange chromatography overview

[@thebumblingbiochemist]

In summary for this background part before I get into the more practical tips: Key points to keep in mind * pH is a measure of free protons (H⁺) which are positively charged, but pH is on a negative log scale so LOWER pH means more positively-charged protons floating around * some parts of proteins can grab onto them - the more there are (lower the pH) the more likely this is ➔ increases charge (an make neutral side chains ➕ charged or ➖ charged side chains neutral) * when pH is high, kiss those H⁺ goodbye! at high pH there aren’t many protons so this is less likely and some protein parts can actually donate them to the cause ➔ decreases charge (➖ side chains can’t get neutralized (they stay ➖) And ➕ side chains have to “give up” their H⁺ (they get neutralized) * proteins have different combinations of parts that give & parts that take * as a result, a protein has an overall charge that depends on the pH
  Each protein has a specific point at which it at which the protein OVERALL is NEUTRAL - there can be ➕ & ➖ charged chains, but they perfectly cancel each other out & we call this charged but neutral condition one of my favorite words: ZWITTERIONIC. The pH at which it occurs the pI or ISOELECTRIC POINT * Above the pI, the protein is ➖ * Below the pI, the protein is ➕    Now that we understand the molecular magic behind ion exchange chromatography, let’s look a bit closer at how we can but this knowledge to practical use.     As I hinted at earlier, there are 2 “flavors” of ion exchange chromatography: ANION exchange and CATION EXCHANGE. Ions are charged things and basically you “exchange” ions from salts (like the Na⁺ or Cl⁻ of NaCl (table salt) with protein ions. Then you can gradually increase the salt concentrations so that those salt ions outcompete the protein and you get another exchange. Or you can change the pH to change the protein’s overall charge (the lower the pH, the more free H⁺ for the protein to latch onto ➔ become more positive & vice versa)    In CATION EXCHANGE chromatography you have negatively charged resin & you’re binding & exchanging positively-charged (cationic) proteins & salt ions. you need to be at a pH below the pI to make sure you’re positive   ANION EXCHANGE chromatography is the opposite - you have positively charged resin & your binding & exchanging negatively-charged (anionic) proteins & salt ions - you need to be above the pI to make sure you’re negative   The more oppositely-charged something is, the more salt it will take to compete it off. And since different proteins have different charges, they’ll come off at different salt levels.  more on protein purification bit.ly/proteinpurificationtech & bit.ly/proteincleaning more on topics mentioned (& others) #365DaysOfScience All (with topics listed) 👉 bit.ly/2OllAB0

[@LionZionOfficial]

Your the Best! 😇❤ Thank you!

[@thebumblingbiochemist]

Happy you find it useful! Thanks

[@meerwasil842]

Thanks mam it was need of the hour ❤

[@llidxenh6561]

a specific protein is functional at a specific pH correct? When you say, you have to make sure the protein is actually cationic or anionic, does that mean at its natural state when its functional? therefore you would try to use that specific pH for evaluating if its below or above the pI? not sure if you answered this in the video but just wanted to clarify. I am assuming you would need the natural pH as well as the pI when you are looking up that info?

[@thebumblingbiochemist]

If you want to study it's functioning, then you will probably want to test it at the pH at which it normally is found. But you can alter the pH a bit (not enough to denature it) to purify it. As long as you know the pI and the pH of your buffer you can determine the charge.

[@llidxenh6561]

@@thebumblingbiochemist Thanks. love your vids. They are helping a lot. Trying to transition into this field.

[@thebumblingbiochemist]

Thanks! So happy I could help. Best of luck!