His-tagged Protein Purification

[@radfatcorgi9384]

such a tiny column, that's adorable)

[@Travelleronearth1]

There should be actual movement seen either in bag or solution in the dialysis set up for effective dialysis.

[@tremeramcbean3719]

I came here to comment that but I see that you already did 6 years ago

[@theomniaplayer3167]

This song is very beautiful!

[@yifanhu1546]

i like desalting column, but not dialysis tube or ultra-filtration tube, because it is faster and usually the recovery rate is higher.

gee, why do you hate your dialysis tube so much?

[@michaella5110]

helpful video! Thanks

[@adarshguptak]

Don't you agitate the cell lysate (or protein extract) with Ni-NTA for binding? Another question, should we incubate the resin in elution buffer before collecting the fractions? Thanks all.

[@EchelonIV]

No need for agitation if you bind by flowing the supernatant through the resin, unless of course if you know that you have poor binding. I usually incubate the resin for 10-15 min before collecting the first set of fractions. Sometimes it's not good to do if you have a lot of bound protein in the resin, as you're essentially incubating your protein in a low volume of high imidazole that can cause precipitation.

[@HannahTheMovie]

Why does the histidine residue bind to the metal ions?

[@Francescar30]

It is because Histidine and Nickel ions have high affinity for each other

[@adarshguptak]

Imidazole moiety of Histidine residues have high affinity to transition metal ions like Nickle, Cobalt, etc.

[@9010444141]

@andezac : to minimize the imidazole concentration from elution step.

[@kashafseducationalsite363]

thiis video is not complete....

[@xzdf366]

damn, I'm on the right side of KZbin again! nice work

[@andezac]

why do you run a dialysis?

[@cowboycatranch]

To get rid of imidazol because it interferes with protein quantification and also because it is not good for your protein to keep it in a buffer with a high concentration of imidazol.

[@Xgjigzigzyixiy]

Narrate this dude

[@xuli3623]

what is the volume of elution buffer for each concentration?

[@nimrakhan8033]

😀😀😀😀 best