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Western blot troubleshooting tips

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

No signal


The primary antibody and the secondary antibody are not compatible.

Use a secondary antibody that was raised against the species in which the primary was raised (e.g. if the primary is raised in rabbit, use an anti-rabbit secondary).

Not enough primary or secondary antibody is bound to the protein of interest.

Use a higher concentration of antibody; incubate longer (e.g. overnight) at 4°C.

There is cross-reaction between the blocking agent and the primary or secondary antibody.

Use a mild detergent such as Tween 20 or switch blocking reagent (i.e. commonly used blocking reagents are milk, BSA, serum or gelatin).

The primary antibody does not recognize the protein in the species being tested.

Check the datasheet or perform a BLASTp alignment to see whether your antibody should react with the target protein; run the recommended positive control.

There is insufficient antigen.

Load at least 20-30 µg protein per lane; use protease inhibitors; run the recommended positive control.

The protein of interest is not abundantly present in the tissue.

Use an enrichment step to maximize the signal (e.g. prepare nuclear lysates for a nuclear protein, etc.).

There is a poor transfer of protein to membrane.

Check the transfer with a reversible stain such as Ponceau S; check that the transfer was not performed in the wrong direction; if using PVDF membrane make sure you pre-soak the membrane in methanol then in transfer buffer.

Excessive washing of the membrane.

Do not over-wash the membrane.

Over-use of the primary antibody.

Use fresh antibody as the effective concentration is lowered upon each re-use.

The secondary antibody is inhibited by sodium azide.

Do not sodium azide together with HRP-conjugated antibodies.

Detection kit is old and substrate is inactive.

Use fresh substrate.



High background


Blocking of non-specific binding might be absent or insufficient.

Increase the blocking incubation period and consider changing the blocking agent, we recommend 3-5% non-fat dry milk, BSA, or normal serum for 1 hr at room temperature. These can be included in the antibody buffers as well.

The primary antibody concentration may be too high.

Titrate the antibody to the optimal concentration, incubate for longer but in more dilute antibody (a slow but targeted binding is best).

The incubation temperature may be too high.

Incubate membrane at 4°C.

The secondary antibody may be binding non-specifically or reacting with the blocking reagent.

Run a secondary control without the primary antibody.

Cross-reaction between blocking agent and primary or secondary antibody.

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

For phospho-specific antibodies: milk contains casein which is a phospho-protein; the phospho-specific antibody will detect casein present in the milk causing high background. Use BSA as a blocking reagent instead of milk.

The washing of unbound antibodies may be insufficient.

Increase the number and time of washes.

Your choice of membrane may give high background.

Nitrocellulose membranes are considered to give less background than PVDF.

The membrane has dried out.

Care should be taken to prevent the membrane from drying out during incubation.



Multiple bands


​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 protein sample has multiple modified forms in vivo such as acetylation, methylation, myristylation, phosphorylation, glycosylation etc.

Examine the literature and use an agent to dephosphorylatem, etc. the protein so that it runs at the expected size.

The target in your protein sample has been digested (more likely if the bands are of lower molecular weight).

Make sure that you incorporate sufficient protease inhibitors in your sample buffer.

Unreported novel proteins or different splice variants that share similar epitopes and could possibly be from the same protein family are being detected.

Check the literature for other reports and also perform a BLAST search; use the cell line or tissue reported on the datasheet.

Primary antibody concentration is too high - at high concentration multiple bands are often seen.

Try decreasing the concentration of the primary antibody. Run a secondary antibody control (without the primary).

The antibody has not been purified.

Try to use affinity purified antibody. This will often remove non-specific bands.

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 protein target may form multimers.

Try boiling in Laemmli buffer for 10 minutes rather than 5 minutes to disrupt multimers.



Uneven white "spots" on the blot


Air bubbles were trapped against the membrane during transfer or the antibody is not evenly spread on the membrane.

Make sure you remove bubbles when preparing the gel for transfer. Incubate the antibodies while agitating.



Black dots on the blot


The antibodies are binding to the blocking agent.

Filter the blocking agent.



White bands on a black blot (negative of expected blot)


Too much primary and/or too much secondary antibody.

Dilute the antibodies more.



MW marker lane is black


The antibody is reacting with the MW marker.

Add a blank lane between the MW marker and the first sample lane.



Band of interest is very low/high on the blot


Separation is not efficient.

Change the gel percentage: use a higher percentage for small proteins and a lower percentage for large proteins.



Smile effect on the bands

Migration was too fast.

Decrease the voltage while running the gel.

Migration was too hot (changing the pH and altering the migration).

Run the gel in the cold room or on ice.



Uneven band size in lanes probed for the same protein


Gel has set too quickly while casting and the acrylamide percentage is not even throughout the gel.

Review the recipe of the gel and the addition of TEMED to the gels, add some 0.1% SDS in water to the top of the migrating gel while it sets to stop it from drying.



Uneven staining of the gel


​Contamination from bacteria

Keep antibodies at 4°C and use fresh buffers covering the gel.

Not enough antibody volume

Make sure the membrane is covered with the antibody and incubate while agitating.


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