Coupling magnetic beads-antibody - Pb in coupling antibody to beads (Feb/13/2010 )
I am trying to couple my Flag antibody (M2 from Invitrogen) to the magnetic beads Dynabeads-protein G or protein A (from Invitrogen too).
I am washing three times my beads with PBS/BSA 5mg/ml before adding 2 to 7 microgram of antibody 2 hours or overnight at 4C on the wheel. Then, I wash twice my beads with PBS/BSA and add my lysate.
My IP didn't work and I detected my protein of interest in the flowthrough after the 2 hours or ON IP. I decided to test my beads and collect the flowthrough after the coupling beads-AB and found out that my antibody is there. I do not understand. I have done this in the past and worked really well. Do I miss something ?
You are saying that the ab is not being bound by the Protein A or G magnetic particles after reacting for several hours or overnight at 4C with end over end mixing but this ab did bind to the same beads previously?
yes I was using the same antibody (in another lab and in another country, but shouldn't change anything !)
Do you have any idea what is going on ?
Rather than waste your ab can you test your methodology using inexpensive purified human IgG or other non-specific ab that is supposed to strongly bind to the labelled particles? This would confirm all the buffers, pHs, and particles are correct and working before jumping in with your expensive ab.