For those not familiar with expression with p. pastoris yeast: proteins are secreted into the growth medium.
This is the medium: 1% yeast extract, 2% peptone, 100 mM potassium phosphate, pH 6.0, 1.34% Yeast Nitrogen Base, 4 × 10-5% biotin, 0.5-1% methanol
Lately I am experience loss of the nickel beads as they turn grey or white after incubating the beads with the medium. The beads are added to the medium and mixed with a magnetic stirrer.
I have done this type of purification about 15 times over the past 3 or 4 years and only lately I am experiencing this problem.
Dear Jason
Sincerelly used IMAC to purify protein from bacterila (gram positive) and mammalian (Expi 293) but never Pichia.
Looking to your medium composition i have 2 main doubts:
- the maximun amount of methanol tollerated from the resin.
- the pH of you sample. Optimal pH for protein binding to imac is 7.5-8 and your buffer is 6 and at the beginning of the culture but wiich is the pH at the end of the culture? 6 or the Pichia change it?
Did you load directly the centriguged media into the coloum or you regulate the ph and or mix it with some buffer?
of course if you do not change bead type and culture condition theoretically it not explain why previuosly your protocool work and now not, but i'm just try to make some hypotesis.
good luck
Manuele
Hi there,
How sure are you about the release of Nickel ion from the beads?
Apart from this I think the best to fully control the composition of your supernatant is to perform diafiltration/ultrafiltration prior affinity purification.
Recommendations:
1. If not already doing, perform TFF of media followed by buffer exchange prior to binding to Ni-NTA.
2. Buy Nickel Chloride (or sulfate) and recharge your column. Super cheap and quick.
3. If possible, ensure that you are only using one column per protein to avoid cross-contamination. Or perform a batch binding and discard the resin after each use. The price per milliliter of resin is quite cheap these days but you can recover and reuse.
Best of luck!
Well, filtration sounds like a good idea but is a bit overly complicated and inconvenient to solve this problem. I have no idea what was causing the problem, but using chemical resistant Ni beads ( PureCube 100 INDIGO Ni-Agarose ) did finally solve the problem.
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