Western Blots on media - (Jan/30/2006 )
Hi, I want to run a time course experiment where I do westerns on the media of my cells after 5 time points. I would like to know how much media i need to remove after each time point in order to see a signal. I am aware that this is dependant on the particular proteins I am looking at but I would like to get a general idea of how much I should take and whether or not i shoudl concentrate the media after i remove it.
Would you please tell us what you are looking at?
So that we can tell you how much you need to remove per time point.
what you can do is to do a try : pick all media and precipitate proteins by TCA acetone or column if you can afford it. quantify the prot concentration you have and do as you need....
I am looking at expression levels of Interleukin 6 and pentraxin (both of which have fairlyhigh mRNA expression in this cell type). I cannot take all the media at each timepoint and then refresh becuase the cells are being treated with an inhibitor and i do not have enough of it to replenish each time. I would like to take data for 3 things:
Real time pcr for IL6 and pentrain from lysate
western for IL6 and pentraxin from lysate
Western for L6 and pentraxin from media after 1, 2, 4, 8, 24 hrs
for the real-time, I would use 24-well dishes and take 5 timepoints + 1 negative control, each in triplicate
I think you will need separate wells for your time points. I do a lot of stuff involving both westerns and real-time with various treatments and timepoints of cultured cells, and I find that trying to conserve too much does you no favors if you want data you can trust (it's more expensive to repeat the whole thing because you were trying to save money) and everytime you mess with the wells to add and remove media and reagents you may be interfering with expression patterns and signaling as well.
for the westerns, if your protein is expressed at very high levels (and not all lost in some post-translational processing and degradation events) then I would think maybe 48-well dishes with use of concentrators?