Davebrid at 16:27, 27 October 2009

2009-10-27T16:27:57Z

← Older revision		Revision as of 16:27, 27 October 2009
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	*Probe blot for >4h with DIG-probe, wash and use secondary at 10 000 X dilute in 5% SMP		*Probe blot for >4h with DIG-probe, wash and use secondary at 10 000 X dilute in 5% SMP
	*Visualize with ECL		*Visualize with ECL

			[[Category: Blotting]]
			[[Category: Protein Purification]]
			[[Category: Protein-Protein Interactions]]

Davebrid: wrote initial page

2009-10-27T16:26:38Z

wrote initial page

New page

==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

Preparation of DIG Labelled Probes - Revision history

Davebrid at 16:27, 27 October 2009

Davebrid: wrote initial page