protein concentration and Pull down
Posted 04 January 2013 - 11:18 AM
I have another question for you
I am going to set up a pull down assay using magnetic beads invitrogen.
In my previous experiment I performed a GST pull down experiment following a protocol inherited by my ex colleague.
Comparing this protocol, especially when I compared the concentration of proteins in the cell extract that people report on their papers, I noticed that I used a concentration of proteins which is too high ...sometime over then 6mg/ml of proteins whereas in the papers people report not more then 1-2 mg/ml.
My question is ..
Which is the criteria to use a correct amount of bait protein coupled as well as the protein concentration in the cell extract ?
Posted 04 January 2013 - 12:43 PM
Posted 04 January 2013 - 02:34 PM
and what about the cell extract ?
I don't think that in this case, using a concentration of protein either 1mg/ml or 8mg/ml the result is the same
it's true that there are a lot of variabilities like the affinity of my protein with the bait, if it's just one protein to pull down or more then one.
Probably the best way could be to make a test with different concentrations of cell extract protein
Posted 05 January 2013 - 01:10 AM