Two-night primary antibody incubation? - (Jan/10/2015 )

Hi,

I'm trying to look for phosphorylation of ATM and Akt (on two different blots).  I know that ATM is not expressed too highly in 184A1 cells, and the phospho-Akt (S473) in my IR-treated cells did not appear differentiated from null treatment (but still showed bands) at 2 hours post-irradiation (I am testing 1 hr in case the phosphorylation cycles off at an earlier point.  The literature is mixed between early (30-60 min) phosphorylation events and later (>3 hr) phosphorylation events.  As for why this is not a time-course experiment, that will eventually be done, but for now, I'm using the same lysate as was generated for a HAT assay for TIP60, which was previously shown in other cells to be done at 40-60 minutes post-IR, so I figured it might be more important to get TIP60 when it is active.  But I digress...).  I woke up with a bothersome cough that I've had for a few days, and it's also quite chilly, and it being a Saturday, I figured I should try to rest it off.  However, I also see mixed reaction as to whether a primary antibody incubation can be done reliably for two nights.  I figure that with the lower levels of ATM normally expressed and the lack of a quality differential signal generated last time with phospho-Akt, a longer exposure period may actually help.  But I am also concerned about whether the antibody would degrade with the longer incubation period or even if non-specific interactions would increase to the point of having an extremely noisy blot.  Can I safely continue incubating my blot at 4 C for another night?

I have done 1,5 days before. The bands were quite sharp.

Not a problem at all, done hundreds of times. With some antibodies, you may get higher background, but most of the time, not, especially if you have done the pre-block right.

Some considerations if you plan a long incubation (rather than it happening due to happy hour hangover)

Make sure things don't evaporate during extended incubation

You can add sodium azide (0.05%) to prevent microorganism growth and consequent antibody degradation.

You can supplement the incubation with fresh dose of antibody next day, done that too (may increase noise)