Preparation of DIG Labelled Probes

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  • 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