Dear Gianluca Cioci

are you working with suspension HEK293 cell culture?

The simples but most expensive suggestion is move to Expi293 expression sistem which is based on a serum free media and guarantee very high expression levels

you can find some information about it on the following link

https://www.blogger.com/video.g?token=AD6v5dyTuEwDXD2ztCnLIFHkG06cCp1jaRuUHOGFtHdiTFnj-yS_TyCyPqzvi6RogJaSP_sXqbJnMDIYplkXODpfOc21BETPPh74J7FJuwMPxHW4X79mURcAl7-V0ApA8BQrGUAgcSc

alternativelly you can try to:

1) Optimize the current purification protocol. e.g how long did you wash the coloumn with lower imidazole buffer before more to 50mM imidazole step and which buffer did you use as binding buffer?

2) Which Nickel resin are you using? You can try to use the Excel resin

https://www.cytivalifesciences.com/en/us/shop/chromatography/resins/affinity-tagged-protein/ni-sepharose-excel-histidine-tagged-protein-purification-resin-p-06009

which is suggested to be optimized for mammalian cultures.

3) You can try to adapt your cells in the expi293 expression medium

https://www.thermofisher.com/order/catalog/product/A1435103?SID=srch-srp-A1435103

as well as in the Freestyle 293

https://www.thermofisher.com/order/catalog/product/12338018?SID=srch-srp-12338018

(which is cheaper bur less performant) and use it for protein production..

good luck

Manuele

Cibacron Blue Dye can be used to deplete albumin from samples, and there are commercial kits that do this. Alternatively you can titrate imidazole into the binding buffer, hoping to knock off the BSA before your tagged protein. Good luck!

Cibacron Blue F3G-A

MARS

Proteominer beads

Hi Ismail,

Have you tried growing your cells in Serum Free Media?

If you can, it is the best way to deal with the issue of BSA.

In my experience,any kits that are used to deplete BSA may also remove some of the secreted proteins from your media.

Good luck, Hediye.