Protocol Online logo
Top : Forum Archives: : Protein and Proteomics

Western Primary AB - (Jan/11/2005 )

Hi! I do some westernbotts. Is it more recommendable to incubate the primary antibody at 4 °C overnight or 60 min at room temperature?



I really can't recommend one way or the other. I would think that this is a matter of try and error. I use the 1h RT method myself most of the time with most antibodies, and get good results from that. A collegue of mine incubates his westerns at 4°C ON a gets also good results. So, both ways may work, but when in doubt, try it! When using commercially available Abs, have a look at the manufacturers datasheet and try what's recommended there first.



I incubate overnight or 2 hours at room temperature with good results. I have never incubated for 1 hour, but it seems to work too