Have you tried to titrate the antibodies? Find the minimal dilution of the secondary that doesn't give you too much background and the highest concentration of the primary antibodies that gives you a signal that is specific enough.

If you want to detect phosphorylated Akt, casein probably won't work, as it contains lots of phosphorylations.

Also check if your BSA is clean enough. 1% m/v might be enough, possibly increase Tween (mininimum 0.1% m/v). Using Tween only in PBS with at least 0.9% NaCl might worth a try, too, to find out, if the BSA might be the problem.

How pure are your antibodies? How have they been made? BSA is sometimes used as carrier protein for peptides in immunizations, so they would need to be antigen affinity purified and then absorbed against BSA during manufacture, but then still a background against BSA is possible.

Hello Fabian Recker

Using higher concentration of antibody can give you high background. Usually primary antibody dilution should be in the range of 1:1000 to 1:5000. The primary antibody dilution of 1:500 could be high. Also, your secondary antibody dilution should be in the range of 1:15000 to 1:20000.

Increase the blocking time to 1.5 hours if needed. Use 5% BSA for blocking.

Increase your washing steps and use the low speed Orbital Shaker during washing.

Best.

Agree with Malcolm Nobre I personally had experienced high background and that was because of the wrong dilution of secondary antibody. so I will suggest checking out the exact recommended dilution of antibodies on the company page from which you have bought the antibodies(type the antibody code number in a browser and will show you all the details), you will find the recommended dilution for WB and try it.

Good luck