Preparation of DIG Labelled Probes
From Bridges Lab Protocols
Revision as of 16:27, 27 October 2009 by Davebridges (Talk | contribs)
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)
Protocol
- 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
