Membrane proteins, owing to their highly hydrophobic nature, have always posed a daunting challenge to biochemists and structural biologists working on the characterization of these "naughty" proteins.
1.5-50µL applied to SDS-PAGE and run at standard conditions.
2.Transfer in wet or semi-dry systems under standard conditions.
3.Use commercial non-fat milk block the membrane, it should be dissolved freshly, centrifuged 10,000 rpm for 10 min, and filtered to reduce background.
4.Incubation with first antibody 2 h at room temperature or overnight at 4°C in 1% blocking solution or PBS/TBS only. The antibody preparation should be centrifuged before use (10,000 g 5min.) to remove any precipitates, and it can be slowly filtered through a 0.45um filter by hand. Optimal working dilutions and incubation time will need to be determined by the end user. Typical dilutions with membrane preparations are 1:500-1:3000 usually.
5.Wash 3 x 5 min. with 50 mL of TBS-0.1% tween 20. Typically the blot should float in the wash solution, and often rocking the blot and wash solution on a shaker helps in reducing non-specific backgrounds.
6.Incubation with the secondary antibody, diluted appropriately 2h at room temperature. Remember that secondary antibody concentration determines the sensitivity of the signal; thus optimization of its final concentration can greatly influence the ultimate signal to noise ratio observed.
7.Wash 3 x 5 min. with TBS-0.1% tween 20 , do not let the blot dry out.
8.Detect the result.