Hello all!

I've been doing WB for a while and have obtained great results for >15 kDa proteins. Now I'm searching for a 13kDa protein, which I do find, but bands are strange and linked one to another (pic).  In same membrane GAPDH (37kDa) is perfect.

I was using pre-cast gels 4-20% but since results were odd I decided to cast them myself: I've done a tris-glicine 12,5% to get a greater separation of smaller proteins using 40% acryl/bis 29:1. Separating and stacking buffers were as usual but i've made new ones for this issue (I use distilled water and no milliq). I'm running also as usual 80V (stack) and then 120V, but I've also tried to run at different voltages like 80V the whole time. Results are always the same for this particular range.

 Another issue I've considered is the samples concentration that are different. I'm loading 15ug/well (I've started with 30ug) prepared with laemmli 4x and volumes range from 8 to 20ul. I don't know if this can be a problem, but like I said proteins > 15kDa are ok.  

Any help is appreciated!