Preparation of DIG Labelled Probes

Materials

DIG (Roche cat # 1 333 054): Dissolve a couple of flakes in 20uL DMSO. This should be good for at least 6 months.
Anti-DIG antibody (Roche cat # 1 207 733)
PBS, pH 8.5
Protein for Labelling (Must not be in Tris, cannot have any free primary amines other than protein, we dialyze protein into PBS before labelling)

Add 350uL of PBS and 25ug of protein to an eppie.
Add 2uL DIG/DMSO solution and mix by gentle inversion
Incubate at room temperature for 15min
Stop reaction by addition of 100uL of 1M Tris to quench.
Dialyse as follows:
- 1h into 1L of 25mM Tris at Room Temp
- 4h into 4L of PBS pH 7.4
- O/N into 4L of PBS pH 7.4
Dilute into 25mL of TBS with 2mg/mL BSA and 0.01% azide
Prepare blot as for a western blot but we block O/N in 5% Skim Milk Powder at room temp.
Probe blot for >4h with DIG-probe, wash and use secondary at 10 000 X dilute in 5% SMP
Visualize with ECL