Detection problems in western blot

Problems with transferring proteins to the membrane

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

• Check the transfer was successful using a reversible stain, such as Ponceau S, before immunostaining.
• If the proteins have not transferred effectively, 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.

Check 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.

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

• Try imaging the blot again with a longer exposure time. This may require some optimization to get right.

• SDS in the sample buffer conveys negative charge to the protein.
• Orient the transfer towards the positive electrode/terminal.
• Refer to the transfer apparatus manufacturer's protocol for correct sandwich layering/direction.

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.

• Add a higher concentration of primary antibody.

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

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

• Run a positive control.

• 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, use lower antibody dilution, higher sensitivity ECL substrate).

• Make sure you use fresh primary and secondary antibodies for each experiment; the effective antibody concentration is lowered after each use.

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.

• Use a new antibody with the immunogen located in the protein of interest.
• Run a protein amount panel with a wide dilution range of the antibody to find the correct signal.
• Contact scientific support for immunogen details.

• Remake a recombinant protein or over-expression lysate to avoid immunogen and tag being positioned at the same end. Alternatively, use a new antibody with an immunogen not located close to the tag.

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.

• 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.

• Note that phospho-specific antibodies may react with a milk blocking agent due to the presence of the phosphoprotein casein. If using phospho-specific antibodies, block with BSA instead of milk.

• Increase the blocking incubation period and consider changing the blocking agent.

  We recommend blocking 3–5% non-fat dry milk, BSA, or normal serum for 1 hr at room temperature.

The temperature may be too high.

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

• Ensure all lysates/samples are kept on ice.

Not enough washing between steps.

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

• Wash extensively in the buffer between all steps.

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

Too much substrate (if using enzyme-conjugated antibody).

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.

• Replace it with a milder substrate.

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

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

• Always keep the membrane clean.

• Make sure to place them back to back.

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 shorter exposure time.

Your choice of membrane may give a high background.

Nitrocellulose membranes generally give less background than PVDF

• Nitrocellulose membranes generally give less background than PVDF; consider using a nitrocellulose membrane instead if high background persists.
• For fluorescent western blot, consider using a low fluorescence western membrane (PVDF).

• Use a stronger lysis method (eg 1%SDS hot method) to get a stronger signal and reduce nonspecific bands.