Here are possible causes and solutions:
- Detection step missed or detection reagents not working: The detection step is not included in the instrument protocol. After the instrument protocol is complete, perform the detection step using your standard detection reagents and protocol manually. Make sure the detection reagents are functional.
- Insufficient incubation with detection reagent: Remove the blot from the detection reagent when signal-to-noise ratio is acceptable.
- Incorrect order of reagents added: Be sure to add the reagents in the order described on page 13 of the manual (https://tools.thermofisher.com/content/sfs/manuals/benchPro_4100_man.pdf) to ensure all steps are performed correctly.
- Poor or incomplete transfer: Make sure transfer apparatus and membrane sandwiches are assembled correctly. Use appropriate transfer times. After blotting, stain membrane to measure transfer efficiency.
- Protein of interest ran off the gel: Use positive control and/or molecular weight marker to match gel separation range to size of protein being blotted. After blotting, stain membrane to measure transfer efficiency.
- Sample was too dilute: Load a larger amount of protein onto the gel.
- Poor retention of proteins or protein weakly bound to membrane: Ensure that transfer buffer contains 10-20% methanol. Use membranes with appropriate binding capacity.
- Inactive or overly dilute primary or secondary antibody: Determine antibody activity by performing a dot blot. Increase antibody concentration as necessary.
Find additional tips, troubleshooting help, and resources within our
Protein Electrophoresis and Western Blotting Support Center.