Hi Nick,

if you need a quick solution now, maybe you can try to bind your protein on an ion exchange column first and then elute in biotin free buffer. If you don't know if anion or cation exchange is better just use two columns in tandem so it should be bound by one of them (adjust the pH to a pH at least 1 Unit from the pI of the protein) . You would save time compared to concentrating and dialysing the biotin. I hope that helps.
Best regards

Hüseyin

Imre Berger says:

IBA says you can efficiently pre-deplete with neutravidin, then use a Strep column to capture your protein.
We never tried this though so I can not comment on this.

Otherwise - why not TWO tags? One oligo-his (pre-purification) and then Strep. TAP - tag basically.
That we do and it works.

Andrea Lages Lino Vala says

Hi Nick,

You can ask invitrogen to produce any media you like with specific components omitted. That is, you could ask for F17 or Freestyle without biotin. If that affects cell growth you can add it back yourself – which may allow you to get away with using less biotin.

Adding avidin works well, but becomes expensive for larger cultures. For buffer exchange, we have an akta cross flow and tried it for buffer xchange but it really only works for small scale cultures... for larger cultures hollow fiber concentration/diafiltration is a much better (and faster) option (spectrumlabs for instance).

Double tagging is well worth considering as Imre pointed out !

Cheers

Sabine Suppmann says:

Dear Nick,

Thomas Schmidt, the Strep „expert” recommends to dialyze cell culture media to remove biotin which is always included in media ( and of course not disclosed) or precipitate the secreted protein with Ammoniumsulfate prior to Strep purification.

And also as Andrea mentioned you need a concentrated sample for efficient binding,.

Best

Sabine

Dear Nick

Hi All,
If my calculation from Joop's (very useful) data  is right then we have approx 2µmol/litre which is roughly 0.5mg. To that we would have to add 60mg of avidin worth about €150 to each litre!!OK luckily we have some data on this protein so I can do a cation exchange to concentrate and buffer exchange. With other proteins we are not so lucky.
Hmmm...OneStrep is beginning to look less attractive/less useful by the minute!
Thank you all for your input-time to get out my S column
Nick

P.S. Why is it so difficult to get this information from e.g. Invitrogen. We only want the concentration of one component and I'm not exactly going to be able to reverse engineer Freestyle F-17 from that info!!
 

Dear Nick,

Thank you for the informative call;-)

We tested the Freestyle F17 medium for Biotin content and also checked the information with Invitrogen (they say 0,7mg Biotin/ Liter Medium).

We measure: 1L FreeStyle F17 medium contains ~0,4mg Biotin =1,4µmol

I hope this information helps;-))

Hi Nick,

I would suggest something similar than Hussein: use an ion exchange column to bind your protein; you can load them at high flow rate, are cheaper, and you are not afraid to ruin them. After loading, you can wash the column at the maximal conductivity that wash impurities and do not elutes your protein (you eliminate the biotin in this way). Next step is to put your affinity column on tandem with the IEX, and increase the conductivity to elute your protein from the EIX. Wash with same buffer till low OD. Next: disconnect both columns, and finally elute your protein from the affinity column. I used this strategy many times when the affinity resin is very expensive or when there are interferents to the affinity column, or when I have to load high quantities of medium to the column (10 or more liters), so you need a quick capture column.

Mario