Protein extraction from cell culture media
Posted 17 February 2009 - 12:55 PM
Posted 17 February 2009 - 03:44 PM
Posted 18 February 2009 - 12:32 PM
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.
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
Posted 18 February 2009 - 03:33 PM
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.
Posted 18 February 2009 - 04:22 PM
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.
Posted 18 February 2009 - 09:27 PM
Posted 19 February 2009 - 03:39 PM
Posted 21 February 2009 - 02:07 AM
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??
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.
Posted 13 March 2009 - 09:14 PM
Posted 16 March 2009 - 04:24 AM
It is better to use ELISA kit to detect secreted proteins
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.