I have been working on western blots for about a month now using actin and fetuin-b primary antibodies to no avail. My background is always high and I only had one good result. I want to know if there is a certain blocking buffer I should use instead of 5% milk with my membranes to get a better result.
I have only used milk for blocking a couple of times but it was always beaten by bovine serum albumin (BSA), casein, or BSA/casein.
My standard blocking buffer is 1 - 5% BSA in tris-buffered saline with 0.1% tween20. I have also used the Western Breeze kits from Invitrogen and in my hands their blocks and alkaline phosphatase detection reagents were very reliable. The preservatives that they include in the stock reagents also allow the block to be reused for up to a week if handled and stored appropriately; keep everything clean, use up to 4 times, and store @ 4 degrees when not in use.
Some antibodies will require specific blocking buffers to perform well.
Background can come from many sources so if you have background with both primary antibodies you should check for other issues as well. Background is a very general term and can be caused by many things, a few I can think of are;
- Overheating during transfer
- Primary/secondary antibodies binding to the membrane.
- Primary/secondary antibodies binding non-specifically to the proteins.
- The interaction of detection reagents with buffer components (phosphate and alkaline phosphatase, tween)
When I was working with actin antibodies I used 2% horse serum in PBST to block and to incubate with antibodies. Or just washed membrane really good from 5% milk in TBST(like 5 times 5 minutes in TBST) because goat antibodies doesn't like milk. But I incubate with milk for 45 min-1h. You can also wash 3 times 5 minutes with TBST after secondary antibodies and then 2 times 5 minutes with TBS. It really helps.