Western blot troubleshooting tips | Abcam (2024)

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  • Western blot resources

    • Western blot protocol

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                        • Anti-Rabbit IgG (HRP) (ab205718)

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                                    Solve your western blot problems with these troubleshooting tips, covering common causes of no signal, high background, multiple bands, and more.

                                    Download the complete western blot guide.

                                    Contents

                                    Detection problems

                                    • No signal or faint bands
                                    • High background

                                    Unexpected or multiple bands

                                    • Low molecular weight
                                    • High molecular weight
                                    • Various molecular weights

                                    Unusual gel or band appearance

                                    • Spots or smudges
                                    • Bands appear very low or very high
                                    • Bands are misshapen or uneven
                                    • Bands appear white (if using ECL detection)

                                    Detection problems

                                    No signal or faint bands

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

                                    Western blot troubleshooting tips | Abcam (7)

                                    Ifall 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. Familiarize yourself with the protocol and check the common pitfalls below.​

                                    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.

                                    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.

                                    Western blot troubleshooting tips | Abcam (8)

                                    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.

                                    Back to top

                                    High background

                                    Western blot troubleshooting tips | Abcam (9)

                                    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.

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                                    Unexpected or multiple bands

                                    Multiple bands at a low molecular weight

                                    Western blot troubleshooting tips | Abcam (10)

                                    Possible causes

                                    Solution

                                    Proteases may have digested the protein.

                                    Add protease inhibitors to prevent protein degradation.

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                                    Multiple bands at a high molecular weight

                                    Western blot troubleshooting tips | Abcam (11)

                                    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.

                                    Back to top

                                    Multiple bands at various molecular weights

                                    Western blot troubleshooting tips | Abcam (12)

                                    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.

                                    Back to top

                                    Unusual gel or band apperance

                                    Spots or smudges

                                    Black dots or speckled background

                                    Western blot troubleshooting tips | Abcam (13)

                                    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).

                                    White spots or smudges

                                    Western blot troubleshooting tips | Abcam (14)

                                    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.

                                    Back to top

                                    Bands appear very low or very high

                                    Bands very low

                                    Western blot troubleshooting tips | Abcam (15)

                                    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.

                                    Bands very high

                                    Western blot troubleshooting tips | Abcam (16)

                                    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.

                                    Back to top

                                    Bands are misshapen or uneven

                                    Smile effect on bands

                                    Western blot troubleshooting tips | Abcam (17)

                                    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.

                                    Bands are uneven

                                    Western blot troubleshooting tips | Abcam (18)

                                    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.

                                    Back to top

                                    Bands appear white (if using ECL detection)

                                    Western blot troubleshooting tips | Abcam (19)

                                    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.

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                                    Western blot troubleshooting tips | Abcam (2024)

                                    FAQs

                                    What are the common errors encountered in western blotting? ›

                                    Even though the procedure for western blot is simple, many problems can arise, leading to unexpected results. The problem can be grouped into five categories: (1) unusual or unexpected bands, (2) no bands, (3) faint bands or weak signal, (4) high background on the blot, and (5) patchy or uneven spots on the blot.

                                    Why is my western blot not working? ›

                                    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.

                                    What happens if you load too much protein in Western blot? ›

                                    Loading too much protein leads to signal saturation in western blots, yet too little produces weak signals. This protocol describes an assay development experiment to determine the appropriate protein load for target and control detection prior to performing the actual western blot experiment.

                                    What causes ghost bands in the western blot? ›

                                    The appearance of white bands ("ghost bands") on blots is usually caused by an intense localized signal, that completely exhausts the ECL reaction with a quick burst of light. Therefore, there is no light produced during development, and a white band occurs. This can also happen with prolonged incubation.

                                    Why is it important to remove air bubbles in a western blot? ›

                                    Air bubbles trapped between the blotting paper, gel, and membrane hinder protein transfer and cause “bald spots” (areas of nontransfer) to appear.

                                    What is the failure rate of Western blots? ›

                                    According to a report on GEN, 41% of researchers admit that their Western blots are unsuccessful at least 25% of the time. Yikes! Western blotting (WB) is a widely practiced analytical technique to detect target proteins within samples using antigen-specific antibodies.

                                    What happens if you transfer too long Western blot? ›

                                    If your transfer is too long, you will detect protein on the membrane that is farthest from the gel. This is more likely to occur to the small proteins first so pay particular attention to where the lower molecular weight ladder bands should appear.

                                    How can I improve my western blots? ›

                                    1. Determine the best usage of the target protein and primary antibody. ...
                                    2. Keep up the protein transfer efficiency. ...
                                    3. Anticipate the effect of gel thickness in Western Blot. ...
                                    4. Make sure to equilibrate membranes and gels on transfer solution. ...
                                    5. Cleaner blots with the right blocking solution. ...
                                    6. Optimize your incubation time.

                                    Can Western blot test be wrong? ›

                                    Since the Western blot test checks for antibodies, it may give false negatives for both conditions if it's administered too soon. In some instances, certain conditions such as lupus may also cause false positives to occur.

                                    How much protein to add to a WB? ›

                                    Load equal amounts of protein into the wells of the SDS-PAGE gel, along with a molecular weight marker. Load 20–30 μg of total protein from cell lysate or tissue hom*ogenate, or 10–100 ng of purified protein.

                                    How to prevent protein degradation in western blot? ›

                                    To improve western blot accuracy, samples should be quickly harvested in ice cold buffer (neutral pH) and should be immediately frozen in liquid nitrogen and stored at −80 °C until use to avoid proteolytic degradation by endogenous proteases (as sometimes indicated by gel streaks) [10].

                                    Can too much protein bind you up? ›

                                    A high-protein diet can result in constipation, especially if you do not eat enough fiber. Protein fills you up, which can naturally decrease your carbohydrate and fiber intake. Other side effects of eating too much protein include bad breath, abdominal pain, dehydration, fatigue, and weight gain.

                                    Why are there so many bands on my western blot? ›

                                    Multiple nonspecific bands on the blot may be due to antibodies of poor quality or at too high a concentration, insufficient blocking, or nonspecific binding due to the presence of SDS.

                                    Why is my western blot all black? ›

                                    The blocking step is critical for preventing non-specific reactions and inadequate blocking can result in a high uniform blocking. However, blocking reagents can also be incompatible with certain antibodies leading to a black blot.

                                    Why is my western blot fuzzy? ›

                                    If you have run the gel at a high voltage for a short period of time, the proteins may not run in a uniform manner, causing blurry bands. Blurry bands can also occur if a mistake is made during the preparation of the running buffer, or if bubbles were trapped during the protein transfer stages.

                                    What are the false negatives in Western blotting? ›

                                    False negatives may be caused by physical loss of a conformational epitope or by post-translational modifications that mask the epitope. Building a successful assay is then down to the scientist, and those pesky laws of physics!

                                    What are the pros and cons of Western blotting? ›

                                    Advantages of Western blotting include the ability to identify specific target proteins, while disadvantages include difficulties in reproducibility and time-consuming procedures.

                                    What is the disadvantage of Western blot test? ›

                                    One main disadvantage of WB is that the secondary antibody may bind nonspecifically to other proteins, particularly immunoglobulin chains, in the extract. A potentially powerful application would involve combination of ELISA and WB methods as shown in a recent study (Carlino et al., 2010).

                                    What are the precautions of blotting techniques? ›

                                    Tips for Successful Western Blotting Transfer

                                    In both types of transfer systems (tank and semi-dry), extra caution should be taken to prevent introduction of air bubbles anywhere between the filter paper, gel or membrane. Thicker gels or larger proteins may require longer transfer times or increased field strength.

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