Immunoprecipitation and 2D gels - (May/25/2004 )
I am attemting to identify the Oestrogen receptor(ER) after immunoprecipitating the receptor from cell lysates and runnning the sample on a 2D gel. I stain the gel with coomasie blue so that any spots that appear contain enough protein to be analysed by MALDI/TOF mass spec.
This means that I need large amounts of protein to begin with, in order to see spots in the area of the gel that I would expect to see the ER. I have such a large amount of cell protein that I need a large amount of ER antibody and therefore a large volume of agarose beads.
To seperate the proteins from the beads I then add the 2D denaturing buffer so that i have enough to put 350microlitres of the buffer and sample on to the centre of the isoelectric focusing channel. I do not pipette up the beads because they disturb the isoelectric focusing, which means there is a lot of sample that I can not access around the beads even when I have spun the beads right down in the centrifuge.
My question is, how can I access this remaining sample which is at the bottom of my tube, surrounding the agarose beads. Is there some way of seperating the beads from the remaining buffer and denatured sample proteins??? I would very much appreciate any suggestion please.
Stick a small piece of whatman1 paper in your tube. Then leach out your sample with buffer from the filter. Good luck