immunoprecipitation problems - (Jul/07/2006 )

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I have been doing IPs for a transiently expressed nuclear protien which is HA tagged. initially there was no such problem but now the Control IgG and anti-HA ab IPs have similar westrn patter
i.e.
1.the Ab is picking up a ladder of bands from 67 to 30 kDa in both IPs (and i am using the same ab for IP and western)
2.the efficiency of the HA-protein pulled down seems very bad .
3.i am unable to decide whether the problem is of degraded HA-protein or Degraded anti HA antibody or a non-specific binding to protein-A sepharose beads.

please can anyone help me out

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what do you mean by control IgG and anti-HA ab IPs.
I just want to be sure I understand well what are your controls.

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Well, i am proceeding with half the amount of cell lysate for an IP using Rabbit anti-HA polyclonal Ab, and another half of cell lysate for an IP with either pre immune serum or commercially available purified Rabbit IgG that should not show any nonspecific binding of HA-tag protein under the similar expt conditions.

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Hi
I'm experiencing the same problem. Does anyone know what's wrong?
/C

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well Cilla i figured out that my problem was due to using Santa Cruz Anti HA polyclonal for the western. The Ab picks up a good expression pattern when it comes to cell immnostaining for confocal analysis so it binds the HA motif alright. But when you do an IP with one Ab i guess it is wisr to use another ab to detect it on Western blots. when i used the sample from the same experiment for a western blot using a monoclonal Ab from Santa cruz, again, i get the impression that all is well in the experiment too. so i did a pull down with Polyclonal and detection with a monoclonal and that's how i have been managing. Also multi banding with anti Ha comes when probably the protein soup loaded has very low levels of the Tagged protein i.e your cells have not got transfected well.
hope my anecdote helps, all the best

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