Western blot troubleshooting tips

Possible causes

Solution

Problems with transfer of proteins to 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 (see diagram).

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.

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.

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.

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

Possible causes

Solution

The primary antibody and the secondary antibody are not compatible.

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 isn't present.

Run a positive control.

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

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).

Overuse of antibodies has reduced their effectiveness.

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.

Possible causes

Solution

The secondary antibody may be binding non-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.

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.

Blocking of non-specific binding may be insufficient.

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.

Incubation temperature may be too high.

Make sure you incubate samples at 4°C. 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.

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

Dilute the substrate and reduce substrate incubation time.

Signal amplification may be too high (if using a signal amplification technique).

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.

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

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.

Possible causes

Solution

Proteases may have digested the protein.

Add protease inhibitors to prevent protein degradation.

Possible causes

Solution

The protein may form multimers.

This is likely if you see extra bands at high molecular weights that are 2x or 3x the weight of the expected bands.

Some proteins will form dimers, trimers, or larger multimers due to disulfide bond formation if the samples are insufficiently reduced.

To prevent this, try boiling the sample for longer in Laemmli buffer during sample preparation.

Possible causes

Solution

The protein may have multiple isoforms or post-translational modifications

Many proteins display bands at slightly higher molecular weights than expected due to post-translational modifications (PTMs) such as phosphorylation and glycosylation or alternative splice variants.

Check the literature to see if multiple bands are reported.

To confirm the extra bands are due to PTMs, you may break down modified proteins by treating samples with suitable reagents. For example, PNGase F can remove glycosylations. The additional bands should then disappear when running another blot.

The cell line may have been passaged too many times.

Cell lines that have been frequently passaged gradually accumulate differences in their protein expression profiles

Go back to the original non-passaged cell line and run these samples in parallel.

The bands may be non-specific.

Where possible, use blocking peptides to differentiate between specific and non-specific bands. Only specific bands should be blocked (and thus disappear).

The antibodies are not purified.

Some antibody formats are relatively impure and may contain additional proteins.

If possible, use antibodies that have been affinity purified.

Possible causes

Solution

The blocking reagent has clumped together, and antibodies are binding to it.

This binding will appear as dots of positive signal.

Filter the blocking agent.

The gel or reagents are contaminated with bacteria.

Make sure you use fresh, sterile buffer (eg our sterile PBS).

Possible causes

Solution

Air bubbles were trapped against the membrane during transfer.

Bubbles will appear as uneven white spots.

Make sure you remove any air bubbles caught between the gel and the membrane during transfer.

You can do this by lightly pressing down on the stack with a small roller.

You should be able to see any bubbles after checking the success of the transfer with Ponceau S.

Possible causes

Solution

The gel has been run for too long.

Before proceeding with blocking and immunostaining, check the transfer of proteins to the membrane with Ponceau S.

If all bands appear very low, you may have left the proteins too long to migrate through the gel.

Try running a gel again for a shorter time before proceeding.

Not enough acrylamide in the gel.

The bands may be very low on the blot if there's not enough acrylamide in the buffer.

This is because the proteins do not experience enough resistance, so migrate too quickly across the gel.

You should generally run lower molecular weight proteins in gels with a higher percentage of acrylamide.

Check this table for suggested gel recipes, and increase the amount of acrylamide if necessary.

Possible causes

Solution

The gel has not been run for long enough.

Before proceeding with blocking and immunostaining, check the transfer of proteins to the membrane with Ponceau S.

If all bands appear very high, the proteins may not have had enough time to migrate across the gel.

Try running the gel for longer before proceeding.

Too much acrylamide in the gel.

The bands may be very high on the blot if there's too much acrylamide in the buffer.

This is because a high acrylamide density can block effective migration of proteins through the gel.

You should generally run higher molecular weight proteins with a lower percentage of acrylamide.

Check this table for suggested gel recipes, and reduce the amount of acrylamide if necessary.

Possible causes

Solution

Voltage may have been too high during migration.

If the voltage is too high, migration will occur too quickly.

Check the protocol for the suggested voltage and decrease if necessary.

Gel may have been too hot during migration.

If the temperature is too high, the pH of the buffer may be slightly altered, which could affect migration.

Run the gel at 4°C: on ice or in a cold room.

Possible causes

Solution

The gel has polymerized unevenly.

When the gel has not polymerized properly, bands can appear wonky or uneven. In extreme cases, lanes probed for the same protein can appear at different molecular weights (see image above).

Check your gel recipe to see if you've added the right amount of TEMED. See our suggested gel recipes here.

Ensure the gel is covered entirely in buffer while it is setting.

Possible causes

Solution

Primary and secondary antibody concentration may be too high.

If the antibody concentration is very high, then the substrate is consumed very quickly. This means very little light is absorbed at this point, leading to a white band when you image the blot.

Dilute the antibody to its optimal concentration.