dot blot - Problem (Jul/25/2010 )

Hello everyone

I would like to ask you if it's necessary a denatured sample to performe protein dot blot? I have that doubt because many dot blot protocols don't mention it.

I've been doing dot blot to detect SCD-1 from rat liver homogenized without results. I have to denature samples???

Thanks

I´m not an expert on dot blotting but I think it depends on the antibody. Is it a commercial one? Because manufacturers usually tell in their data sheets whether the antibody works for native or denatured protein or both. Did someone else in your lab already use this antibody for western blotting, immunocytochemistry or something else? Because then you would have a hint which conditions to use. I would suggest to try both in parallel (native and denatured protein extracts), although sometimes denaturaration buffer components may cause some dot-spreading on the membrane.