His-Tag Protein Purification theory & practice - Immobilized Metal Affinity Chromatography (IMAC)

IMAC (Immobiolized Metal Affinity Chromatography) is a way to help purify proteins that we’ve added a “His tag” onto to make them specifically sticky to resin (little beads) that are coated in a metal such as nickel (Ni) or cobalt (Co). So when we express a protein with a His tag then break open the cells (lyse them) and flow all that stuff that was inside the cells (lysate) through it, our protein will stick, other proteins (without the tag) won’t, then we can get our protein to unstick - all alone!
The sticking works (hopefully) by the metal acting as a go-between connecting the resin and the protein (specifically the chain of Histidine (His) amino acids we’ve attached to the end), unlike some other resins where the protein interacts directly with the beads. So with IMAC you have bead:metal:His-tag where those : are something called coordinate covalent bonds, which are where an atom with a lone pair (like that N in the His chain) shares a pair of electrons with a metal.
And these bonds are reversible - which is good for your protein being able to come off - but not good if it comes off to easily - and not good if the metal comes off the beads. So instead of just binding in one place, you want something that “bites” the metal multiple times - and we call such molecules that can form multiple coordinate covalent bonds CHELATORS.
So you have a chelator (like NDA or NTA (more below)) “PERMANENTLY” stuck to the beads. And then that chelator clamps down around the metal. And then the string of Hises you’ve attached to your protein acts as a chelator that bites down around the metal too.
When metals bond to nonmetals, they often do so as COORDINATE COVALENT BONDS - these bonds form when one atom shares 2 electrons with a metal ion in the middle (in normal covalent bonds each atom shares one) - we call the result a COMPLEX and you can learn more about them here: bit.ly/2BDBml0⠀
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In order to complex with a metal, an atom has to have a lone pair of electrons they can share - like NITROGEN!!!!!. If a molecule has more than one atom with a pair to spare, they can “bite down” on the metal in multiple places - POLYDENTATE. We call such multiple-toothed-biters CHELATORS⠀
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His’ N’s can act as electron pair donors in coordinate covalent bonds, so His is able to take a bite. And this can hold metals in place in the active site of enzymes, where those metals can give & take electrons to help promote reactions & stabilize awkward reaction intermediates. ⠀
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If you have a lot of His in a row, it’s like having a polydendate (many-biting) thing - one of those chelators. So if something has a string of His it can bind to metal tightly. And we can take advantage of this to help us purify proteins. ⠀
In protein chromatography, we can use different types of resin to separate based on different properties - properties that are “inherently normal” to the protein - like size or charge or something we’ve added on through recombinant expression trickery. AFFINITY CHROMATOGRAPHY uses resin that recognizes something really specific - usually an affinity tag we’ve added onto the end of the protein (by putting the genetic instructions for it before or after the instructions for our gene in the plasmid). Because affinity chromatography is recognizing something “unnatural” and highly specific, it can (hopefully) remove most contaminating proteins - but not all.⠀
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A common affinity tag is a His tag, which is just 6 or 8 Histidines. Other proteins have Histidines too. But not that many in a row, so your protein will bind preferentially. It’ll hog the column and the other proteins will flow through.Then you need something that will outcompete the His tag to get your protein off. Bring on the imidazole. It looks like His, so you can flood the column with imidazole to push the His-tagged proteins off. But before you flood it, you wash it with low levels of imidazole to remove non-specific binders that are just binding cuz they happen to have a lot of Hises.⠀
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So you go from ⠀
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resin:Ni:imidazole⠀
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resin:Ni:His-tagged protein⠀
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then resin:Ni:imidazole + His-tagged protein⠀
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In the beginning, when you’re loading your column, flowing through all that gunk, your protein is able to push off the imidazole because Imidazole mimics a single His so can only take a single bite whereas your tagged protein has a lot of them. When you are ready to push your protein off (once you’ve washed off all the other stuff) you can do it with imidazole, but you have to add a ton of it since your protein’s a better binder - you have to go with the “flood it out” strategy. more here: bit.ly/histagpurification

[@George-kw7qr]

Thank you so much! I learned much more from your videos than from my biotech professors :)

[@thebumblingbiochemist]

Happy to help!

[@kittyrodriguez8902]

I only just started this video to brush up on some technicalities for work, but I really like the way your explain and teach. I've done this style of explanation with other students I had to train in my previous lab and it makes understanding processes so much more buildable and understandable from the get go. Thanks!

[@thebumblingbiochemist]

Thank you so much! Best of luck with your work!

[@Cooking4me_928]

Amazing information. Thanks

[@Reree-gz5bg]

Finally got around to subscribing and love your videos

[@thebumblingbiochemist]

Thank you!

[@amirah4034]

Hello foreigner. Your explanation is very well. I learn

[@thebumblingbiochemist]

Thanks! Glad to hear that!

[@danirurajapaksha7990]

Hi ma'am thank you so much for the video . I am hoping to study biochemistry and I just finished my A levels here in Sri Lanka. Could you please do like lecture series of biochemistry. People would love that. I know that you are busy with your post doc. Please consider. Your notes/lecture slides are very beautiful and really easy to understand. Keep up your good work best of luck

[@thebumblingbiochemist]

Thank you so much but I can't do a comprehensive lecture series. Thanks for understanding and your very kind words. Best of luck with your studies!

[@junan1279]

Hi! Your content is well explained! Nice job! Do you know for GST tag purification, why do we use reduced glutathione and not oxidized glutathione? What's the purpose of washing with PBST and PBS?

[@thebumblingbiochemist]

Thank you! Reduced glutathione is the substrate for GST, not the oxidized form, so GST will bind to the reduced form and that's what you want to use. PBST has detergent, whereas PBS doesn't. Detergent can be used to help reduce nonspecific interactions and/or to increase protein solubility. Hope that helps

[@claudyomande2686]

Thank you! I'm learning a lot from here and many of your videos! How do you determine optimal residence time (for sample load) for his-tagged protein to be bound to Ni resins? Is it truly protein specific or just the amount of His that may affects the binding?

[@thebumblingbiochemist]

Thanks! Happy I could help! It can vary from protein to protein and also as a factor of the total volume because you need to take diffusion into account

[@ahmedalhusays3097]

I appreciate what you are doing, Do you have an idea about TurboID ? is it like His-tagged protein?

[@thebumblingbiochemist]

Thanks! TurboID is a totally different thing. In that case you're trying to figure out what proteins are binding to, rather than purifying a protein. But you do use a form of affinity capture to capture the biotinylated proteins. I don't have any posts on that sorry!

[@aziranafisahamiruddin313]

Hi! thank you for the video! very helpful. For the regeneration of the column, can i replace NiSO4 with Nickel (II) Chloride?

[@thebumblingbiochemist]

Yes - that should be fine. Good luck!

[@mssapphins]

Thanks for the video! Do you know why Ni or Co are preferred for His-tag purification but not Fe-III?

[@thebumblingbiochemist]

Thanks! I'm guessing it has to do with the coordination and number of sites: www.goldbio.com/articles/article/his-tag-metal-affinity-cations-whats-the-difference-again

[@rasonsong9854]

where did you get the double ending things of the tube? 0:35

[@thebumblingbiochemist]

dunno sorry! the lab just had them

[@emantaher1551]

Thanks Can I use the same method for chitin colum?

[@thebumblingbiochemist]

A similar concept but different resin & competitor.