Biotechniques | Basics of Making His-Tags & Nickel Affinity Chromatography

As always, the steps of putting a gene insert into vector/plasmid AND nickel affinity chromatography depend on your specific lab and protocol. Here, we look at the general theory of engineering a poly-His tagged protein and Ni affinity chromatography and what they are used for in the lab.

[@giftymammen1515]

Wow, whatta explanation. I've been searching for the literature for hours and have been pounding my brain ever since on this topic. The minute I landed onto this video, all my confusions are gone, crystal clear now. Thankyou so much ❤️😭

[@satinderjit4]

4th year bio-chem major here and this was very helpful in understanding the basic idea.

[@robertadu-agyei7616]

so insightive

[@AL-eh3jx]

I've been searching for hours and finally got to watch your video. This helped me a lot in my lab! Thank you!

[@CatalystUniversity]

Glad this was helpful.

[@AL-eh3jx]

@@CatalystUniversity I have a question and would appreciate your help! If we use Nickle binding buffer containing imidazole( low concentration) before the incubation with resin, would any binding between the His-tagged protein and Ni ions happen at this stage?

[@yourfuturedocburenbeiya]

This is so helpful ; thank you for posting this video doc! We're using it in lab to isolate dmAANATA and I couldn't find the molecular weight but came across this video. :)

[@CatalystUniversity]

No problem! And thanks.

[@qamarhennawi9137]

Wow, this is an awesome explanation; I've been seeking for something like this for a decade!!!!, Thank you very much.

[@vikingmakesballs7799]

Abdala Covid vaccine brought me here

[@christinamrad7341]

what if a protein contained histidin in the sample and is not our protein of interest?

[@ying2694]

How about the antibiotic resistance gene?

[@uzoechisamuel]

Vector + Gene = Plasmid

[@snobbips5107]

Awesome explanation sir😊👌

[@bioscienceswithshahtareenswati]

Amazing 👏 🙀

[@NehaSharma-kp8op]

Thank u so much for making this vedio with ausome explanation

[@CatalystUniversity]

You're very welcome!

[@jenna9992]

in the gel analysis, what are the weights on the molecular ladder?

[@CatalystUniversity]

To be perfectly honest, it depends on your ladder. If you know the company and the exact product, the company website will have this available. For example, if I knew I was using the 1 kb DNA Ladder made by New England Biolabs (very good company, by the way), I could type into google search "New england biolabs 1 kb ladder". Long story short, you would need to find the exact product or at least know the size of the ladder (e.g., 1 kb).

[@davalcom]

Thanks a lot man

[@karl-leopoldkontrus6544]

important to say, that the his-tag does not undergo post-transcriptional modifikation , or did I miss something? VERY HELPFUL VIDEO!!

[@pingr.8730]

This is very useful! Thanks for uploading this video. It's exactly what I was looking for 😊💙

[@khurshedakabirov8671]

Excellent explanation. It would be great if you added little details about how to make the protein (induction, SDS, etc...).

[@ignacioquijadag.7917]

Buen video bro, lastima que entendí la mitad porque no sé ingles jajaja pero estuvo muy bueno

[@khaledkourbane9182]

Good good job ...thank you Dr Amazing biology informations

[@xuehanxu8814]

Very well explained. Thank you so much!

[@faizaarshad705]

what would be the procedure, if I want to introduce his tag protein into a cyanobacteria

[@balasubramanianb2107]

When wash with salt solution that nickel binded to NTA is removed or not sir?

[@carlostettey2889]

it will be great if you can show how to design the primers for creating these vectors.

[@saherk5021]

Thank you so much for such a nice and simple explanation

[@robertstadler9897]

Very good explained! Thank you and keep going.

[@uzoechisamuel]

wonderful lecture. Thanks.

[@khaledkourbane9182]

Thank you for those inforamtions ...

[@jimmyyoung1657]

Clear interpretation for a newer to this experiment.

[@izzatiauni3721]

It is really helpful!

[@CatalystUniversity]

Thank you!

[@titoflash1212]

Thanks a lot again Kevin!!!

[@AA-gl1dr]

Thank you so much!