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Detection problems in Western blot

Explore possible causes and solutions to various detection problems in Western blot.

All bands, including the ladder, are faint or have no signal

If all the bands on your blot including the molecular weight ladder are difficult to see, it could indicate a problem with your technique rather than the protein you’re trying to detect. 

Possible causes

Solution

Problems with transfer of proteins to the membrane.

Protein transfer may be ineffective due to incorrect direction of transfer or inaccurate prepartion of the membrane.

  • To see if your transfer was successful use a reverasble stain, such as Ponceau S, before immunostaining.

  • Check the transfer was performed in the right direction.

  • If using a PVDF membrane, make sure you pre-soak the membrane in methanol and then in transfer buffer.

Too much washing between steps.

Washing with buffer between steps is necessary, but sometimes washing too aggressively can remove detection reagents.

  • Reduce the duration or number of washing steps.

The wash or incubation buffer is contaminated with bacteria.

Bacterial contamination can come from many sources in a lab, such as through spores in the air or small stray fibres.

  • Use fresh, sterile buffer (eg our sterile PBS).

Reagents may have lost activity due to improper storage and handling.

Always check and follow the storage instructions for your products on the datasheet.

  • Avoid excessive freezing/thawing.

  • If using fluorescent detection, the fluorophore may have been damaged by too much light exposure. Store and handle fluorophores and fluorophore-conjugated antibodies in the dark and minimize light exposure by wrapping the vial in foil.

You may have used the wrong filter settings for detection.

  • Ensure you set the instrument to read the correct wavelengths.

There may not be enough exposure time when imaging the blot.

Exposure time can take some optimization to get right.

  • Try imaging the blot again with a longer exposure time.

Bands in the sample lanes are faint or have no signal

If only the sample lanes are difficult to see, and the molecular weight ladder is unaffected, this suggests there are issues detecting the protein of interest.

Possible causes

Solution

The primary antibody and the secondary antibody are not compatible.

Secondary antibodies should be carefully matched to your primary antibody across different characteristics. If you're struggling to find the right secondary antibody, read our guide on selecting a secondary.

  • Make sure you use a secondary antibody raised against the primary antibody species.

  • Make sure that the isotypes of the primary and secondary are compatible.

Not enough antibody is bound to the protein.

  • Add a higher concentration of primary antibody.

  • Incubate the sample for longer with the antibody (eg overnight) at 4°C.

The protein of interest is not present.

  • Check the scientific literature to see if the protein is expected in your cell line.

  • Run a positive control.

Not enough protein is present.

  • Make sure you load at least 20–30 µg protein per lane, use protease inhibitors, and run the recommended positive control.

  • Use an enrichment step to maximize the signal (eg prepare nuclear lysates for a nuclear protein).

Antibodies used have low effectiveness

Effective antibody concentration is lowered after each use, therefore it is best to avoid using antibodies across experiments whenever possible.

  • Make sure you use fresh primary and secondary antibodies for each experiment.

Buffers may be incompatible with the detection method.

Some buffers contain reagents that may interfere with detection. For example, sodium azide is an inhibitor of HRP, so it is unsuitable for use with HRP-conjugated antibodies.

  • Check your buffers don't contain any incompatible reagents, and change the buffer if needed.

High background

Possible causes

Solution

The secondary antibody may be binding non-specifically

Even when matched against the species and isotype of your primary antibody, some secondary antbodies may not bind specifically.

  • Run a control without any primary antibody.

  • Make sure you use a secondary antibody raised in a different species to your sample.

  • Try a secondary antibody that has been pre-adsorbed against the lg of the species of your samples.

Primary antibody concentration may be too high.

  • Dilute the antibody further to its optimal concentration.

The secondary antibody may be binding to the blocking reagent.

Note that phospho-specific antibodies may react with a milk blocking agent due to the presence of the phosphoprotein casein.

  • Add a mild detergent such as Tween 20 to the incubation and washing buffer.

  • If using phospho-specific antibodies, block with BSA instead of milk.

Blocking of non-specific binding may be insufficient.

  • We recommend blocking 3–5% non-fat dry milk, BSA, or normal serum for 1 hr at room temperature. If this does not work, increase the blocking incubation period and consider changing the blocking agent.

Incubation temperature may be too high.

Proteolysis, dephosphorylation and denaturation can all start if samples are kept above 4°C  throughout the protocol.

  • Make sure you incubate samples at 4°C and keep on ice throughout the western blot process.

Not enough washing between steps.

Residual unbound antibodies or other reagents remaining between steps can produce a high background.

  • Wash extensively in buffer between all steps.

  • If using fluorescent detection, be sure to remove Ponceau S before immunostaining as this can autofluoresce.

If using enzyme-conjugated antibody, there is too much substrate.

Secondary antibodies for western blotting are most often conjugated to an enzyme (eg HRP or AP) but can be conjugated to fluorophores or proteins.

  • Dilute the substrate and reduce substrate incubation time.

If using a signal amplification technique, signal amplification may be too high.

  • Reduce the amount of signal amplification (eg conjugate less biotin to secondary antibody if using biotinylation).

The blot has dried out.

  • Prevent the membrane from drying out during incubation by keeping it covered in buffer.

Exposure time may be too high when imaging the blot.

Exposure time can take some optimization to get right.

  • Try imaging the blot again with a longer exposure time and optimize from there.

Your choice of membrane may give a high background.

Nitrocellulose membranes generally give less background than PVDF

  • Consider using a nitrocellulose membrane instead if high background persists.