I've been trying to troubleshoot this western blot for weeks and I need help. I'm blotting for my protein of interest, LOX, and am using GAPDH as loading control. I am able to detect the loading control always on the blot but can't detect the protein of interest. I've tried several different conditions including adding up to 150ug of protein and still no dice.
1. Extract protein in RIPA with protease and phosphatase inhibitors by sonication and incubation on ice for 30mins. (Also tried Protein extraction kit from Full Moon)
2. Load 40-50ug protein
3. 10% SDS page
4. Transferred to PVDF membrane for 1.5 hours
5. block for 2 hrs in 5% milk TBST
6. 1:1000 primary co-incubation (1:1000 GPADH) overnight at 4C (also tried 1:500) in Odyssey buffer
7. wash 3x 5min in TBST
8. 1:5000 secondary incubation for 1 hr at RT in Odyssey buffer with LiCor secondary antibodies
9. wash 3x 5 min in TBST
10: Image with Bio-Rad Chemidoc
I also run a positive control protein sample to the blot (from the same company as antibody) and the antibody recognizes this positive control and I see bands only in this lane.
My loading control always shows up as a nice band but I can't detect my protein of interest. I was thinking the antibody may just be bad but the reviews online and the datasheet kind of argue that the antibody should be pretty good for western blot....
ANY help/suggestions would be greatly appreciated!