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BSA in adipocyte cellculture? Alternatives??

adipocytes BSA starving western blot

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#1 fat_cell

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Posted 22 November 2011 - 12:53 AM

Hi cell culture gods!!
I work with preadipocytes, which were differentiated into mature adipocytes now for some days. My goal now is to test the influence of different treatments on the cells and in particular on a target protein, which will be analysed with western blotting. My problem is, that I have to starve the cells overnight. Usually I would do this with my pure DMEM (high glucose) and 0,1% BSA. Afterwards my treatment will be applied in exactly the same medium (DMEM * BSA) for 24h. But my western blot will not work with such amount of BSA and there is untill now no way to get rid of the BSA without getting rid of my protein as well.
Do you have any ideas what to put in instaed of BSA? Or do you think that the cells will survive only with DMEM for 2 days? Or should I starve the cells with BSA and then use BSA free medium for 24h, would this be surviveable?

Thanx for every answer ,
fat_cell

#2 rhombus

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Posted 23 November 2011 - 08:59 AM

Hi cell culture gods!!
I work with preadipocytes, which were differentiated into mature adipocytes now for some days. My goal now is to test the influence of different treatments on the cells and in particular on a target protein, which will be analysed with western blotting. My problem is, that I have to starve the cells overnight. Usually I would do this with my pure DMEM (high glucose) and 0,1% BSA. Afterwards my treatment will be applied in exactly the same medium (DMEM * BSA) for 24h. But my western blot will not work with such amount of BSA and there is untill now no way to get rid of the BSA without getting rid of my protein as well.
Do you have any ideas what to put in instaed of BSA? Or do you think that the cells will survive only with DMEM for 2 days? Or should I starve the cells with BSA and then use BSA free medium for 24h, would this be surviveable?

Thanx for every answer ,
fat_cell


Dear Fat cell,

This has been a problem for many years and I am afraid that I am going to be of limited help

Researchers have been trying to totally replace FCS/FBS in cell culture but as yet have been unsucessful. There are a range of commercially available low serum media, protein free and chemically defined media's. However the cells need to be wheened of full serum (over a period of weeks) and then onto these alternatives....with differing success rates.

But what you want to do is take your cells and then put them into one of these media's immediately ....this is the problem.

You may get away with protein free and therefore chemically defiend media's for 2 days............however these media's also have pitfalls

Chemically defined normally includes compounds such as Transferrin, Selenium and Hydrocortisone......all dirty compound with many different cellular targets

I would try one of these chemically defined media's.....however beware....the manufacturers normally will not tell you the complete composition of their media.....because then you could make it yourself!!!!

Sorry to be of limited help.


KIndest regards


Uncle Rhombus





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