I want to measure some secretary proteins in the supernatant and I want use conditioned media. I have proceeded in this manner: seeded the same number of cells in the same volume of medium, treated the cells with different reagents (or vehicles) for the same period of time and taken the same volume of conditioned medium to test. I would say it is normalized. Note: IN DMEM I did not add FBS or any antibiotic. Is this method correct?
Plate cells in equal number and allow them to adhere (24 hrs). Serum deprived these cells for 12 hrs or 24 hrs with 2-3 washes. Now add your factor for the desires period of time in medium alone (like DMEM). Collect the spent medium after the treatment time is over and spin it down to remove any dead cells (2-3K rpm). As this spent medium has your protein of interest in a diluted concentration, concentrate the medium in a 3kD cut off membrane tube. After passing it through the 3KD cut off tube, you will get spent medium concentrate which is around 200 micro lit. Do a Bradford to load the equal amount of protein in different treatments. If you want to check the status by western load equal amount of protein in each well and may normalize again with any secreted protein which does not get altered by your treatment. Alternately, before proceeding to blocking (after transfer) do a Panceu s staining to normalize the amount of protein loaded. Now you can check the status of your protein in the spent medium.
Conditioned medium is spent media harvested from cultured cells. follow the link for details http://atcc.custhelp.com/app/answers/detail/a_id/306/~/conditioned-medium
Hi Ramesh,
The whole point of using the conditioned medium is to use it as is and test for the secretory proteins in it. The conditioned medium is generally the supernatant derived from a given cell culture under given conditions. You shouldn't add other reagents to it and then test the reaction. Test the conditioned medium directly for its secretory proteins.
Hi, Ramesh, I agree with previous comments: conditioned medium is a the medium whcih cells adapted for optimal growth. Please, also consider that conditioned medium may contains not only secteedcprotein, but also low molecular weight molecules like aminoacid. Sugar anmd iones contents of conditioned medium may be different form original one. However, aftre prolong cultivation, conditioned medium may be also toxic because of accumulations of dangerous product of cell metabolism. I would suggest you to chdck secretory proteins at differentbtime points: cells may secret different proteins at different culture stages. Good luck!
Yes, I agree with previous comments. Traditional term "conditional media" (CM) is not applicable in your experiment - I presume. Conditioned media is usually spent media from healthy cultures and added to other cultures to enhance growth. Your work appears to be analysing secretary proteins under different conditions. Once you have measured protein levels in these media and then want to study effects of these proteins on other cells, you can then consider these media as CM (for some other cells).
Plate cells in equal number and allow them to adhere (24 hrs). Serum deprived these cells for 12 hrs or 24 hrs with 2-3 washes. Now add your factor for the desires period of time in medium alone (like DMEM). Collect the spent medium after the treatment time is over and spin it down to remove any dead cells (2-3K rpm). As this spent medium has your protein of interest in a diluted concentration, concentrate the medium in a 3kD cut off membrane tube. After passing it through the 3KD cut off tube, you will get spent medium concentrate which is around 200 micro lit. Do a Bradford to load the equal amount of protein in different treatments. If you want to check the status by western load equal amount of protein in each well and may normalize again with any secreted protein which does not get altered by your treatment. Alternately, before proceeding to blocking (after transfer) do a Panceu s staining to normalize the amount of protein loaded. Now you can check the status of your protein in the spent medium.
Ya thank you every one for informative answers. In my case I have to do according to the Imran Khan answer, Because I want to see the expression of 110KDa protein by WB.
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