Here are potential causes for high background and corresponding solutions:
- Too much HRP in the system: Further dilute the HRP-conjugate, see guidelines in Table 1 in the user guide (https://assets.thermofisher.com/TFS-Assets/LSG/manuals/MAN0018858_SuperSignalWestAtto_UG.pdf).
- Inadequate blocking: Optimize blocking conditions.
- Inappropriate blocking reagent: Try a different blocking reagent. Use clear milk or StartingBlock blocking buffer instead of dry milk.
- Inadequate washing: Increase length, number or volume of washes.
- Film has been overexposed: Decrease exposure time or use Pierce Background Eliminator (Cat No. 21065).
- Concentration of antigen or antibody is too high: Decrease amount of antigen or antibody.
- Poor antibody specificity: Use SuperSignal Western Blot Enhancer (Cat. No. 46640).
We also recommend minimizing handling of the membrane, using clean gloves while handling, clean plasticware, glassware, forceps, or other contact surfaces. We also recommend trimming of the membrane to remove excess area.
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.