*From Edmondson DG and Roth SY, 1998, Methods, 15:355.
- Separate the protein sample of interest on an SDS-PAGE or acid urea gel. Transfer the gel to a solid support membrane. Either nitrocellulose or PVDF membranes yield good results. However, the PVDF membranes are easier to handle and give somewhat stronger signals, probably due to increased protein binding capacity.
- After transfer, blots may be stained for 5 minutes in freshly diluted Ponceau S (Sambrook, 1989).
- Destain the membrane in water until the proteins are clearly visible. Light pencil marks can be placed adjacent to markers or identifiable bands to mark them for future reference. The stain fades quickly so the marks must be placed immediately.
- Following Ponceau S staining, block the blots for 2 hours in 0.05% Tween-20 in PBS, followed by 2 hours in 1% BSA in PBS.
The blocking of non-specific binding sites on the membranes is a critical step. Too little blocking results in high backgrounds while extended time in blocking solutions results in weakened or lost signal. The reason for the diminished signal is unclear, but protein renaturization apparently takes place during the blocking step, so an optimal renaturization may require limited blocking.
- After rinsing in PBS, the blots may be used directly for probing or may be wrapped in plastic wrap and stored for up to 2 weeks at 4°C.
- To make the probe, translate the protein of interest in vitro in the presence of 35S-methionine. Dilute the IVT in PBS containing 1% goat serum, 0.3% BSA, in a volume sufficient to cover the membranes to be probed (typically 3 ml). Pre-incubate the blots for 10 minutes in dilution buffer without probe, and then incubate for 2 hours at room temperature in a 50-ml conical tube containing the diluted probe. Rotate the rubes mechanically throughout the binding reaction.
- Wash the membrane 4X in 200 ml PBS for 5 minutes per wash. Background is generally quite low and extended washing does not reduce background substantially.*
- Allow the membranes to air dry, and then expose to film or to phosphoimaging screens. Overnight exposure to X-ray film is usually sufficient to detect positive interactions.
Probes for Far Westerns can be generated by other methods. A recombinant protein can be biotinylated by commercially available reagents and then detected with streptavidin-horseradish peroxidase, or labeled with 125I. Alternately, an unlabeled recombinant protein can be used and then detected by traditional western.
*If there is a background problem, it is generally best to try to purify the probe on a microspin column such as Amicon. After translation, the probe is diluted with translation buffer or with 40mM Hepes, pH 7.4, 40mM DTT, and purified by centrifugation at 10, at room temperature.
Ponceau S Stain
- 2 g Ponceau S
- 30 g Trichloroacetic Acid
- 30 g Sulfosalicylic Acid
- Bring to 100 ml with water
- Dilute 1:10 with water prior to use
- 137mM NaCl
- 3mM KCl
- 7mM Na2HPO4
- 15mM K2HPO4, pH 7.9-8.0