Western Blotting FAQ

Western Blotting

1. How much protein should I analyse?

We recommend analysing a 50ug whole cell lysate (total cellular protein) or a 25ug nuclear extract per lane. 


2. What percentage acrylamide gel should I use to resolve the proteins?

<13 kDa 15% 14 - 50 kDa 12% 50 - 100 kDa 10% > 100 kDa 8% 


3. What membrane should I use?

We recommend using nitrocellulose. Although PVDF and other similar membranes can be used, they can sometimes lead to increased background particularly with goat polyclonal preparations. 


4. What blocking buffer should I use?

We recommend using 1X TBS, 5% milk, 0.05% Tween-20. Blocking should be conducted for 30-60 minutes at room temperature or overnight at 4o. Please note that Tween should be omitted from the buffer if incubating overnight. 


5. What dilution of primary antibody should I use?

Check the datasheet of the individual antibody as this usually notes starting dilutions. If there is not specific information on the datasheet then we suggest that you perform a serial titration against relevant positive and negative controls.