Protein extraction from cell culture media - (Feb/17/2009 )
I am trying currently to find out whetheraa protein is expressed from a cell monolayer. I suspect the protein is sectreted from the cells and exists in the cell culture media. Does anyone have an idea how i can measure the concentration of the culture media and how i can make to run a gel with this sample? I have already attempted the BCA method but since the media i pink i get nonvalid values. I have to get rid of the pink color. Any idea anybody??
Buy some medium that is free of phenol red (the pH indicator in most media). Also worthy of note is that typically medium is used with a percentage of FBS, which will easily swamp the measurement of any proteins you are getting excreted from the cells.
bob1 on Feb 17 2009, 04:44 PM said:
Thx so much for your answer.
By the was i fixed the thing with the red color. I just compared it to FBS free media and i got my concentration. I am not really interested at this point in finding the exact details of the expression. I just need a positive signal in order to go on with the experiment. Why is the phenol red free medium so important? Will this ruin my Western blot? And how do i concentrate my sample?
Thx again so much
The phenol red is the red colour in the medium, not the FCS. FCS will interfere with the ability of the BCA assay (detects all proteins) to detect any secreted proteins, of which yours will be one of many, so it will not be a good measure of how much of your protein is being secreted. I would go as far as to say that you will not be able to distinguish between samples as the measurements will be within the statistical margin of error for the BCA assay. Also how do you deal with cells that have died in the medium releasing all the intracellular contents?
Concentrate by precipitating and re-suspending the protein in a smaller volume. Again, FCS will interfere with this as it will make up the majority of the protein in solution.
you couldnt be more right than that, but my problem is not the total protein content of the sample (FBS or debris from dead cells). In fact so far in my samples i have delt with total protein and i correct my signal afterwards with densitometry. I can still keep the cells overnight or for 24 hours in FBS free media or i can relativate my measurement to FBS free sample in order to let BCA compare (and as a matter of fact i do come up with a valid measurement). Regardless of how delute my sample will be, and it trully is, and apart from the fact that the vast majority of the protein in the sample will be from FBS i just want to do a test and see if there is even a small amount of my protein of interest in there. Even a faint signal will do. So i wonder if i can run a western blot with the sample that i already have (wich means Phenol and FBS containing). Will Phenol cause me any trouble as far as protein running is conserned? Have you ever done sth similar?
If i get the desired signal i surelly will go forward as you already proposed.
If you need to look at the total proteins, try TCA, acetone or ammonium sulphate precipitation. All protocols are readily available off the Net. Then you can run a gel to look for your protein, especially if you use ammonium sulphate to do a differential precipitation.
FCS and phenol red will not interfere with a western blot, but the medium will probably be too dilute to detect the secreted protein of interest. As Swanny says, you will need to precipitate to concentrate your protein levels.
Aris on Feb 17 2009, 09:55 PM said:
If there is a good ELISA for this protein. Then go for it.
Else you could try to speedvac the media and then try to BCA and western.
yobou on Mar 14 2009, 06:14 AM said:
I'm agree, for secreted proteins we use ELISA, but for zimography of metalloproteinases we have run electroforesis without problem with the culture medium which had phenol red and FBS.