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I have now received the beads, and was wondering how much to add of the 50% slurry per immunoprecipitation.
I have read on the label of the beads 100ug (therefore I assume the concentration of the antibody it will be 100ug/200ul gel which is 0.5ug/ul. If I use 20ul of beads gel +20 ul PBS per IP (50%slurry), then I will be using 10ug of antibody per IP (the protein comes from one 6-well plate for each IP). Isn’t this too much? The general protocol of Abcam says to add 70 to 100 ul of the slurry but that would be even more antibody.
I used to use 2ug of IP antibody plus 100ug of protein for IP and worked always fine, so I am wondering if 10ug won’t be too much for one IP and will result in non specific binding?? I guess it is a bit different, because here I am pulling down directly with the anti-“flag” antibody, but would like to know how much antibody I should add to the IP.
Thank you so much again for your help,
Asked on Nov 08 2012
Thank you for your reply.
I would recommend for the first time use of using a couple of different antibody concentrations and then seeing which one gives the best signal. You could have one condition where you use 2ug of antibody and another where you use 5ug of antibody.
If there is anything else I can help you with, please let me know.
Also, since you are based in the UK and I am based on the West coast of the USA, if you wish to receive faster replies, you could email mailto:firstname.lastname@example.org and that will go to our UK team. I am more than happy to give any assistance, I just wanted to let you know why there is a delay in my response.
Answered on Nov 08 2012