**EDIT: 30ug of Protein is loaded measured by microBCA

I've been trying to blot for beta-actin on cell culture protein lysates for weeks without success. The image below is the best I have gotten. It is loaded with ladder, sample 1, 2, 3 twice.

Our lysates are from Bone-marrow mononuclear cells lyzed in RIPA + protease inhibitors and stored at -80. Protein concentrations are read by Pierce MicroBCA.

SDSPAGE: We do SDS-PAGE under reducing conditions in a 4-20% Tris-Glycine mini-gel with the Thermo Mini gel boxes. I run them at 180V for 1h15m. I've attached an image I get after a comassie stain of the gel (Imperial Protein Stain)

Transfer: After washing in dH2O, I transfer onto a PVDF membrane with the iBlot2 dry transfer and easily see the bands on the blot.

Blotting: I then block with 5% BSA (fresh) in TBST for 1h RT, incubate in b-actin antibody (CST, 1:1000) overnight at 4c. I then wash with TBST and incubate with anti-rabbit HRP secondary (abcam, 1:3000) 1h at RT.

Detection: I read the blot by adding the Amersham ECL reagent from GE for 1 minute and capturing on LAS3000 darkbox with 5m exposure.

I have troubleshooted the protein determination assay, the electrophoresis time, transfer time, and secondary antibody. I've not yet tried a new primary or ECL reagent.

What is going wrong??