which step for IP is better? - IP protocol (May/09/2008 )

Hi Guys,

Can anyone tell me which steps gives better results for IP??

(A) Incubate antibody to beads first then add lysate

( Incubate lysate with antibody then add beads


I need a protocol that gives clean bands because the results i'm getting now are not optimal

Thanks

Unfortunately there is no absolute answer. There is no condition that will always work best regardless of the antibody or conditions. Personally, I've had better luck conjugating the antibody to the bead. This way you can also block the beads in BSA after conjugating the antibody but before adding lysate. If you really need to clean up an IP you need to preclear your lysate with IgG control beads. If you are trying to IP with a rabbit antibody, if you preincubate your lysate with control rabbit IgG, you can potentially remove background or non-specific binding.