I'm currently using 10k amicon untra centrifugal filter to do the buffer exchange on IL-2. My IL-2 was bought from here:
https://www.peprotech.com/recombinant-human-il-2
I dissolved IL-2 in 100mM acetic acid for long period storage as indicated by the supplier. But for our downstream experiment, we need to carry out in PBS buffer. So I planned to do buffer exchange using previously mentioned filter. However, the recovery rate is very low, about 10-20%. Some details:
IL-2 concentration before centrifuge: 0.1mg/mL
Buffer used: 1*PBS, or 1*PBS with 5% trehalose
Centrifuge spead and time: 14000rcf 5-10min
No protein was detected in the filtrate
It seems that IL-2 was stuck on the membrane. I tried to use the solution remained in the filter to wash the membrane for several times during centrifuge, but it didn't work. What should I do next? Should I change the filter? Thanks.
Hi there,
Did you actually estimate the content of the starting solution (ie. the initial recovery from the powder)?
You will probably not be able to recover the protein on the filter.
Next time, dry down the amount you need in a speed vac to pull off the acetic acid which is volatile. Then you can reconstitute in PBS. If the IL-2 came dry from the company, it must be stabile to being lophilized, and retain its activity.
The company should have some advice. Try a small amount to test to make sure activity is retained.
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