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

[@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!

[@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!

[@Reree-gz5bg]

Finally got around to subscribing and love your videos

[@thebumblingbiochemist]

Thank you!

[@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

[@Cooking4me_928]

Amazing information. Thanks

[@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

[@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

[@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!

[@emantaher1551]

Thanks
Can I use the same method for chitin colum?

[@thebumblingbiochemist]

A similar concept but different resin & competitor.