⚗Nickel Affinity Purification of His-tagged Proteins || Practical Biochemistry 

Hi dear, 1. The concentration of imidazole for the wash and the elution solutions are pretty standard and you don't have to test it. The wash solution has a little to take off impurities and the elution has a lot to completely elute your protein off the column. 2&3. columns for size exclusion and HPLC and such come already pre-packed and you don't want to mess with their packings(if you do it messes with the seperation) so they must be used with a machine that can pump liquid through them. The packing doesn't really matter for nickel affinity so you can just pipette the resin onto a column and use gravity.

Once the protein is eluted it is in 6M guanidine so this has to be dialyzed out eventually. I take 400ml refolding solution which has arginine and a bunch of other stuff in it (lmk if u need the recipe) and dialyze my 20ml elution in it overnight in cold room or fridge. Then take the dialysis tube and put it in a low phosphate +urea buffer (4L x2) then ur final buffer (4L x2) for me 20mM tris. Do a CD and check if it's folded. If u can't find the buffers online I will list the ingredients for u.

It's 6M GuHCl, 250 mM NaCl, 50 mM sodium phosphate, ph=8 (choose ph so it's at least 1 away from the proteins pI so it doesn't precipitate out)

Yes definitely

Hi. The agarose is in bead form and the nickel is bound to it. It is the same state in the bottle but on the column there is a filter so as gravity pulls everything down it just appears to be condensed and solidified. Once you are finished you can still pipette out the agarose-nickel beads.

@@biochemistmelo ok Mam Thankyou! And Two more doubts.. 1) I was going to do Batch purification, So in this there are chances of getting lost my desired protein during 'removal of flow through before washing step'? 2)My Ni NTA Agarose got expired on Aug-2019, So May I still make it in use? And to encounter both of these problems do I need to use more Ni NTA agarose than recommended?? P.S - Binding capacity of my Ni NTA Agarose (Qiagen) is mentioned 50mg/ml

@@divyanshbhadiar1508 if I was in your situation I would just test the purification with a smaller amount and collect aliquots from each step (lysate, flow through, wash1, final wash, elution) and then run an sds page gel. This will answer all your questions... if the expired resin still works, if your protein binds the resin ... everything. If your purification is under denaturing conditions( + GuHCl) you need to do TCA precipitation before running gel but if not just add loading buffer

Yes. You need to have something like a thrombin cleavage site after your his tag. Then incubate your purified protein with thrombin for 1-2 hrs ( find the right time by running a gel from samples at different times) then pass it through a benzamidine sepharose column to get rid of the thrombin. Add pmsf ( 1ml 100mM) to stop residual thrombin activity. I used to do this but you lose a good amount of protein too so if you don't think the his tag is interfering, it's ok to leave it.

@@biochemistmelo thanks!

Hi I am not familiar with HRP2 but I googled "hrp2 isolatiom and Ni NTA" and this paper came up so I think u can. It seems to be Histidine rich. www.ncbi.nlm.nih.gov/pmc/articles/PMC8522059/

can you send me your producer in PDF form.I have a problem of English language

@@gutemajebe4365 procedure you mean?

@@biochemistmelo yes

If you have a kit, they always come with instructions and you should follow that but the concept is the same. Equilibrate, bind, wash, elute.