Just a couple of comments from what I do:

1. use TBST instead of PBST

2. 10% for blocking is high. I use max 5%

3. secondary Ab should be at 37C for 1-1.5 hrs.

4. Do maybe 5 washes at each step.

5. I also see the protein ladder light up sometimes in the film, that may possibly due to too much ECL.

Just a couple of comments from what I do:

1. use TBST instead of PBST

2. 10% for blocking is high. I use max 5%

3. secondary Ab should be at 37C for 1-1.5 hrs.

4. Do maybe 5 washes at each step.

5. I also see the protein ladder light up sometimes in the film, that may possibly due to too much ECL.

Thanks for the reply. The secondary antibody at 37 seems a tad strange to me, but I'll read more about it and consider your suggestions.

MS

For the secondary antibody i recommend room temperature. The time depends on your detectable protein amount and primary ab dilution, but 0,5-1 hour should be sufficient.

regards

Using your primary antibody ON at 4oC might improve things, otherwise i agree with the previous comments. We always do secondary antibody 1hr RT. 

Thank you Fabian, Tjerk and Raffaella. I am leaning towards using the 5% milk and the secondary antibody for 1 hr at rt. I will try the experiment again on Friday and update you all with my result!

yes this occur some time it is not a big problem,

just see ur prim antibody concentrations, and also as extra care check for some contamination of protein ladder, u can use from another lot and then see what happened. best luck for ur experiments

If you see this trouble occasionally, it would likely be a leaking molecular weight marker from the well of SDS-PAGE gel. Better check the gel after you remove the comb. Or, If you load too much of molecular weight marker solution to small well, this could hapen. Besides above problem, as other answers suggest too high concentratuin of secondary Ab or too long exposure of the blot to the Ab will cause the trouble.

Thanks to all!

could be better to use biotinlated protein ladder