Low Signal or No Signal Troubleshooting in Western Blots

Low signal in western blotting often means poor transfer quality. What are the possible reasons for the poor transfer quality in western blotting and how to prevent weak signal in western blot?

The following are Western Blot troubleshooting guide for easy to solve low signal issues, and can help you get sucessful Western Blotting results.

Western Blot possible causes & solutions for low signal or no signal

Low signal/no signal in a western blot

Low signal / no signal in a Western Blot
Possible cause Solution
Insufficient amount of antibodies present • Increase concentration of primary and/or secondary antibody.
Antibody exposure time is too short. • Increase the exposure time.
Antibodies may have lost activity. • Test antibodies by performing a Dot Blot.
The cell or tissue type does not express the protein of interest. • Perform a positive control, preferably from cell or tissue lysate already verified to express the target protein.
The expression of the protein of interest may be very low. • Sensitivity may be increased by performing an immunoprecipitation prior to the Western blot.
Insufficient amount of antigen present. • Load more protein on gel. Also, if the specific antigen concentration is too low, try enriching the antigen by fractionation or by immunoprecipitation.
Weak Ag expression. • Eliminating Tween during primary Ab incubation may improve Ab binding.
Insufficient antigen binding to membrane. • If the antigen has low molecular mass, it may pass through the membrane. Switch to a membrane with a smaller pore size, or switch to a different type of membrane.
The antigen is masked by the blocking buffer. • Test different blocking buffers, such as milk, serum, BSA in Tris-buffered saline and phosphate-buffered saline. Test different concentrations of each.
Improper sample preparation for gel loading. • Ensure that the protein in the lysate is stable. Use appropriate protease and phosphatase inhibitors, etc.
• Ensure protein samples contain SDS, and have been heated prior to gel loading.
• Include a reducing agent such as dithiothreitol (DTT) and/or 2-mercaptoethanol.
The polyvinyl wrap from certain sources may quench the signal. • We suggest repeating the incubation with chemiluminescence reagents and placing the blot between two pieces of write-on acetate transparency film, and then expose the film.
Excess methanol in the transfer buffer • Too much methanol in the transfer buffer decreases the transfer efficiency of proteins from the gel to the membrane; however methanol aids in protein binding to PVDF or nitrocellulose membranes – a balance is needed.
Air bubbles between the gel and the membrane (leading to "holes" after transfer) • Air bubbles are not conductive and, therefore, interfere with protein transfer – this can be seen as a "patchy" transfer with "holes". The tips and suggestions are to roll over the gel/membrane sandwich with a pipette to drive out any air bubbles, increasing the surface contact between the gel and the membrane; check the sponge pads – if they are flattened, they may not clamp the gel and membrane together with enough force.
Insufficient transfer time or current • Large molecular weight proteins may require a longer transfer duration; A large molecular weight protein may be difficult to transfer from the gel at very low current settings.
Improper cell treatment. • Stimulate cells with the appropriate chemical, protein, etc. Verify that the stimulation works.
Excessive washing of the membrane. • Reduce the number of washes.
Substrate incubation is too short. • Increase substrate incubation time.
Substrate has lost activity. • Test substrate using a positive control.
The detection enzyme may be inactivated. • Sodium azide inactivates horseradish peroxidase (HRP) irreversibly, so do not include sodium azide in any HRP-labeled reagents. Bacterial contamination also diminishes HRP activity. HRP conjugates should be kept bacteria-free, handled with sterile technique and stored under recommended conditions.