Re: seconday and specific Ab concentrations?

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     I routinely stain cells (lymphocytes) for flow with both a primary and secondary Ab for PCNA, cyclins, etc.  (I have also used FITC conjugates for some of these proteins.)  I know little about PARP specifically but the same basic principles (as for the majority of intracellular antigens) should apply.

     "1) Should I perform a separate incubation step for the seconday Ab then the PARP specific Ab; or are they incubated together?"

     No, I recommend two incubation steps after fixation AND permeabilization of your cells:  the first is with your primary anti-PARP Ab and may need to be anywhere from 30 minutes to overnight in duration.  You will need to try a range of incubation times as well as Ab concentrations (see below) to determine the optimal.  The second incubation (with FITC-Ig Ab) can usually be done for approximately 30 minutes to one hour.  (In addition, all incubations in our lab are done at 4 C.)  (For references with protocols using primary and secondary Abs for flow cytometric intracellular protein (cyclin) staining check out the multiple papers by Dr. Z. Darzynkiewicz and colleagues during '94-'97 etc., i.e., Exp. Cell Res. 220: 226-231, 1995.)

     "2) If incubated together, how long? (original protocol was 45 min at 4 C),"

     Again, it is my understanding that this single incubation for 45 minutes at 4 C is really only appropriate for pre-conjugated Abs.  Not recommended for your purposes.

     "3) Next, how do I determine the proper concentrations/volumes for each Ab (the published protocol stated 20 ul [5 ug] of the FITC-conjugated Ab was used)."

     You will have to try a range of both primary and seconday Ab concentrations in order to determine the optimal.  PharMingen recommends 1ug of some of their Ab's per million cells, but I have certainly used less than this to fine effect.  I typically use my primary Ab's at concentrations ranging from 1:15- 1:50 (although I would initially try a wider range than this) and typically use the FITC-Ig at 1:40.  But again, the Ab concentration AND length of primary Ab incubation need to be optimized specifically for PARP.

     With regards to volumes, for both the primary and secondary incubations I have good success resuspending the pellet in 40ul PBS and adding 10ul of the appropriate Ab concentration.  (However, I have also resuspended pellets in as much as 100ul PBS with similar success; just remember that this is a further dilution of your Ab.)  Lastly, I typically dilute the Abs in PBS + 1% BSA + 0.25% Tween 20 (optional), keeping in mind that once diluted they seem to have limited storage life at 4 C.  (I usually make up only what I need at that time from stock.)

     Hope that this helps.

                      Leigh Eward