Client has provided harvest from CHO cell line, expressing recombinant His6 protein.
Last time I fortuitously selected Excel as purification medium (after reading) and purified dirty peak with approx 100 mM Imidazole step, (trailing peak double bump in step - perhaps both dimer and tetramer forms of my protein?? I've not seen this before.
To increase purity, I diluted imidazole out, and repurified over Talon to give sharp cleaner peak (single), then polished on SEC to separate tetramer and dimer forms.
Client, mentioned that they'd successfully used Co-NTA or Ni (no specifics)
Last night I changed my initial protocol and passed 1L of my new harvest (containing no Imid) over straight over Talon 1ml column, to purify under std conditions with 4,8,16,32, 64, 100 % 500 mM Imid.
Sadly today my column was clear and translucent and no longer pink, nothing detected at any wavelength (260/280/254) using Akta pure.
Is it more likely that cheating agents in cell culture media stripped column?
Rather than volume of harvest passed over (1L pH7 1ml/min)?
Can I add cation - Zn or Mg to bind chelator at present at unknown conc.
Does chelator cause translucent appearance? Usually urea ur GuHCl?
Would you recommend Excel, desalt,Talon, SEC
Hi Lesley, Are you referring to Sigma’s His-Trap excel product? (I think the excel refers to the flow rate.) If so your two column matrices have different affinities and recognition of the his . While Talon is a very strong binder it’s recognition of the site is poorest of all the IMAC materials. The fact that Talon is able to bind in absence of competition as you showed in your clean up step shows it’s capable of working in a less complex environment. Looks like you had a good protocol the first time!
Hi Joan, many thanks for the reply.
HisTrap excel is IMAC resinfrom GE Healthcare with the strongest resistance to EDTA (5 mM 10mM 24 hrs).
I don’t think EDTA is a problem in your media. Ni has the strongest affinity. ThAt makes it really good for picking it out of crude. Co and even Zn have higher avidity and are better at separating out bound impurities. Perhaps if you dilute the supernate or pass it more slowly through column giving it more time time to interact you might get some binding. Also don’t neglect the possibility that different batches of column or different pre-washes could affect binding ability.
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