I am looking for a protein secreted by BMDCs over 24h. I need allot of cells (2 * 10^6) so i also need allot of medium (i am using RPMI + 10% FBS (R10)). However, turns out my protein of interest is difficult to detect in the amount of medium I need to use (due to the late time point and number of cells). Is it possible to precipitate the proteins in the supernatant (eg. by salting them out), spinning them down, concentrating them in PBS then running them on a western? Or are there much better ways to concentrate supernatants?
The first thing to be aware of is that the 10% FBS adds a huge amount of protein to the medium, mostly albumin. Removing it before attempting to collect secreted proteins would be advisable. There are resins available for this purpose. Better yet, see if you can get your cells to grow in serum-free medium.
The proteins in the supernatant can be concentrated conveniently using ultrafiltration.
Hallo Einar, deciding on a strategy to concentrate your protein depends on the properties of your protein.
If your protein has a MW < 30kDa, you can do size exclusion filtration. Most major plasma proteins are bigger than 30Kda and you can get great purification using those filters.
If your protein has a MW > 60 kDa, then, as Adam suggested, the best way is to remove albumin and IgG's from your supernatant by using Blue Sepharose and protein A resins, respectively. and then concentrate your protein using size exclusion filters.
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