Thanks.

Here you can see the difference between different milk concentrations. 

For me it looks like the signal intensity goes down due to higher concentrations. But there is less unspecific binding, too.

we use 5% milk for blocking and .5% for primary and secondary.

Once the blocking is done one can reduce the blotto (milK) to 0.5%

the simple reason is that you save on blotto.

We use exactly the same blocking solution for both blocking and antibody incubation.  I think Kunal is correct -- it's just a matter of saving resources.  

I don't change the milk concentration between blocking and primary/secondary solution. It really helps me get clean looking blots with little to no background. The milk also seems to have a stabilizing effect on the antibodies and allows them to keep longer.

However, whenever I am looking for a difficult-to-detect protein (a phospho-protein or one that is at low levels in the cell) I reduce my 5% milk solution to 3%.

Hello

I prepare it at 5% in TBS1x but in some protocols we need to Dilute secondary Antibody in 3% dry milk in TBS 1x to reduce background

Good Luck

https://www.rndsystems.com/resources/technical/troubleshooting-guide-western-blot

Thank you guys for your attention!

I'm trying different milk concentrations for my blots at the moment. I will let you know if i see significant changes in detection quality

 i use 5 % milk in PBS for blocking and 5% milk in PBS(0.1% tween) for antibody dilution.  reducing milk in antibody dilution presumably because the high milk concentration hinders binding of the antibody to the target protein. 

Thanks.

Here you can see the difference between different milk concentrations. 

For me it looks like the signal intensity goes down due to higher concentrations. But there is less unspecific binding, too.

Konstantin, thanks for sharing those blots. It illustrates the principle correctly, that the more crowded the solution becomes with due to milk components, the more difficult it becomes for both specific- and non-specific binding to occur. 

This is also neatly illustrated by switching to BSA in place of milk, or to fish gelatin; in general, the signal strength improves but so does the noise with these two blocking agents.

we used to make blots in 0.5% horse serum in PBS. for FACS and IF staining we use 2% FCS in PBS, I bet this would work for western too, have not tried it though. Its a good way to use up samples of FCS provided by different suppliers, for blocking the batch is not so critical I think.

adding milk to primary antibody (or even secondary) is not a requirement for blotting. However it can work as a blocking and carrier protein to increase the shelf-life of antibody during storage. personally, I prefer serum albumin instead because it contains less fat. If you are not using automated/vaccum blotter then you can add high concentration of milk (up to 5%). Otherwise milk concentrations >>0.2% would block the vaccum filters. obviously, if you intend to use milk in primary antibody then you can reduce the blocking step time accordingly.

Thomas, I first started doing westerns with horse serum in TBS, though I do not remember the exact concentration of serum. The signal was stronger than in 5% milk/TBS, although it also had more background as well. 

I've noticed that antibodies (other than phosphospecific antibodies) that demand the use of BSA as a blocking agent generally do not bind to their epitope as strongly (and are more prone to non-specific binding); using milk with them usually leads to a drastic reduction in signal.  

Konstantin, just to share - I typically start with 5% milk in my antibody solutions (dissolved in TBS). To help preserve them I add 0.02% sodium azide. I have gone as high as 10% milk at times, but rarely.  

Thank you for your answer. Your opinion fits perfectly to my shown results. The stronger and more concentrated the blocking solution the less signal you get. Furthermore you get less background. I think everybody knows what to do now!