concerning the westrn blot ..........I am diluting my antibodies in 5% bsa , but I get very high backgroud though I an diluting the secondry antibody in 5% milk .
My question is . Can I block the membrane in 5% milk and add the primary Ab diluted in 5% BSA ?
You can try using milk first. Block the membrane with 5% milk in TBST for an hour at room temp. and wash.
Then put the primary antibody in 5% BSA overnight, wash again and incubate with secondary antibody in 5% milk for 2 hours at room temp., wash and then detect.
The recommended protocol for most of the antibodies is to use 5% BSA to dilute. Using milk to dilute antibodies can be good if you need to reduce unwanted background, but it can also prevent antigen recognition by the primary antibody, resulting in a weaker signal.
Actually, using milk or BSA will depend on the expression of your protein and the recognition of your antibody. Both options generally work perfectly, but several aspects must be taken into account in practice. Milk contains mainly casein, which, being a phosphoprotein, may interfere with the experiment if antibodies that identify phosphorylated sites or proteins are being used, in addition several colleagues mention that said blockage is more effective if their protein is highly expressed and its antibody recognizes it faithfully. Conversely, if you have a poorly expressed protein and your antibody recognizes certain phosphorylation sites, blocking with BSA is more recommended. Finally, if your situation meets the characteristics to perform the block with milk, I see no problem in diluting your antibody in BSA and incubating it in the membrane blocked with milk. My question would be: is it very necessary to block with milk when the antibody is diluted in BSA? why not just block and dilute in BSA?
There is something preventing you from blocking with BSA?
You can try using milk first. Block the membrane with 5% milk in TBST for an hour at room temp. and wash.
Then put the primary antibody in 5% BSA overnight, wash again and incubate with secondary antibody in 5% milk for 2 hours at room temp., wash and then detect.
The recommended protocol for most of the antibodies is to use 5% BSA to dilute. Using milk to dilute antibodies can be good if you need to reduce unwanted background, but it can also prevent antigen recognition by the primary antibody, resulting in a weaker signal.
For my case, I was using 5% milk in TBST for 15-20 mins for blocking the membrane. For Primary Ab, I have used 3% BSA + TBST for 1 hour at room temp.
You can check with your washing steps.
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