Hello guys! I'm using an Amicon® Ultra-15 Centrifugal Filter Device with a 3 kDa membrane to concentrate my protein, which is a 6,9 kDa protein (about 9 kDa considering Histag) but my protein keeps passing through the membrane.
I tried to concentrate a 0,1% BSA solution to clog some membrane pores but it seems my protein keeps passing through it. Any tips or solutions that could help me?
Ps: 3 kDa is the low MW membrane available for this device
Ps2: I'm concentrating at 5000xg/20 minutes/4ºC
Giovana Casteluci BSA should not pass through as it is about 65 kDa.
Try using BSA alone and do a protein assay of the filtrate before and after. if most are found in the filtrate, then the filter device might be the issue.
Its max operating g (from website)
"When using a swinging bucket rotor, spin the device at 4,000 × g maximum for approximately 15–60 minutes. When using a fixed angle rotor, orient the device with the membrane panel facing up and spin at 5,000 × g maximum for approximately 15–60 minutes."
but if you exceed 5000 g then the filter unit might be defective. rpm should be converted to g force according to specification.
the filtration +/- error can be more than 1 or 2KDa
https://www.merckmillipore.com/MY/en/product/Amicon-Ultra-15-Centrifugal-Filter-Units,MM_NF-C7715
The minimum size is 3 kDa and they tested with a 12 kDa protein, you might want to ask their tech representative regarding this issue.
We used a lot of the Amicon® Ultra-15 Centrifugal Filter Device in our lab. Interesting results you got there.
Putting aside damaged membrane, the size issue is more complex.
When proteins fold they might be much more compact then when they are spread-open. Thus 6 KDa protein (on the gel - with SDS = spread open) could pack in such a way they it will go through a 3 KDa membrane.
Try the BSA, but even whole membrane will not mean good separation. Try a dialysis bag.
https://www.researchgate.net/publication/267929648_Retention_of_modified_BSA_by_UF_membranes
Dear folks, thank you for your answers. The past month was really busy for me.
I read the manual and I am following all the requirements but it seems my protein keeps passing through the membrane. I am using a GE concentrator right now, it works but at a slow pace...I know it is not ideal but it has been the only way to concentrate my protein.
My protein has about 9 kDa when folded so it should not be a problem concentrating it in a 3 kDa membrane.
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