Dot blot protocol

Last edited Tue 1 July 2025

Dot blot is a technique for detecting, analyzing, and identifying proteins. The dot blot technique is a straightforward immunoassay used for detecting specific proteins or nucleic acids. This technique is similar to western blot, but protein samples are not separated using electrophoresis; instead, proteins are spotted through circular templates directly onto the membrane or paper substrate. Dot blotting is a convenient method that does not require protein separation. It is considered a powerful tool and a cost-effective technique for rapid detection and quantification of target proteins. Dot blot uses antibody-based detection, allowing researchers to identify specific proteins and nucleic acids by applying the sample directly onto the membrane.

​The concentration of proteins in crude preparations (such as culture supernatant) can be estimated semi-quantitatively using the dot blot method if you have both purified protein and the specific antibody against it. In this case, compare the signal from the unknown sample to that of the standard and estimate the concentration. Dot blotting can be used to estimate protein levels and target protein concentration, making it useful for various downstream applications.

However, a limitation of dot blotting is that it does not provide information about the molecular weight of the target protein due to the lack of protein separation, unlike western blots and western blotting.

Stage 1 - Sample preparation

Prepare a series of dilutions using a commercially acquired peptide sample (with a purity of >90%). Preparing different concentrations of the sample is essential for accurately determining the target protein concentration. Using a recombinant protein as a standard allows for precise quantification in dot blot assays. It is also important to include suitable controls to ensure reliable and interpretable results.

Select specific antibodies and high-quality antibodies when performing a dot blot to ensure reliable and accurate results. Confirm antibody specificity during antibody selection to guarantee that the antibodies will selectively detect the target proteins. Additionally, determine the optimal antibody concentration for your experiment to achieve precise detection. When available, it is recommended to use recombinant monoclonal antibodies.

Materials required

• Peptide sample
• 1×TBS (20 mM Tris-HCl, 150 mM NaCl, pH 7.5)

Steps

Select the appropriate antibodies for dot blot testing.

Obtain corresponding phosphorylated and non-phosphorylated, acetylated and non-acetylated, or methylated and non-methylated peptides.

Dilute the peptide to the concentrations of 100 ng/µL, 5 ng/µL, 1 ng/µL, 0.1 µg/µL and 0.01ng/µL in 1×TBS as shown in the table below.

Stage 2 - Membrane preparation and sample loading

At this stage, load the sample onto the membrane of our choice. Here we describe the procedures for the two most commonly used membranes: nitrocellulose and polyvinylidene fluoride (PVDF). PVDF membranes are widely used in dot blot assays due to their high binding capacity and suitability for high-quality protein detection, allowing the sample to be applied directly for antibody-based assays.

Materials required

• Your sample

• Nitrocellulose membrane

Steps

Draw a grid on the membrane with a pencil.

• This is to indicate the region you are going to blot.

Load the sample onto the membrane.

• Spot 1 µL of sample onto the nitrocellulose membrane at the center of the grid using a narrow pipette tip.

Let the membrane dry.

Materials required

• Your sample

• PVDF membrane

• Methanol

• 1xTBS

• Vacuum

Steps

Prepare the PVDF membrane for use.

• Separate the PVDF membrane from protective sleeves and activate it with methanol for ~20 sec.

• Wash the PVDF membrane with distilled water to replace methanol.

• Assemble manifold(s) with filter paper cut to size on manifold and activated membrane in the following order from bottom to top: manifold base, filter paper, PVDF membrane, and manifold top.

• Add 100 µL of 1xTBS buffer to each well.

• When ready to load peptide, pull through using a vacuum pump.

Load the sample onto the membrane.

• Load 100 µL of each concentration of peptide per your manifold layout plan.

Leave the membrane for 30 mins, then pull through using vacuum.

Open the manifold by releasing opposing diagonal clips and cut the membrane as required.

Number blots and note the locations of dots on the membrane for imaging orientation.

Stage 3 - Blocking

Blocking is required before antibody incubation to reduce signal from non-specific sites on the dried membrane. Blocking prevents non-specific binding and reduces the chance that proteins or antibodies bind non-specifically to the membrane, which can interfere with signal clarity and accuracy. It is important to optimize the antibody binding conditions during the blocking step to achieve clear and specific results.

Materials required

• Prepared membrane

• Blocking buffer (example: 5% milk in TBST or 2% BSA)

• Wash buffer (TBST: 0.1% Tween-20 in TBS)

Steps

Block the membrane for 1 h at room temperature on a shaker.

Wash the membrane three times with wash buffer.

• 10 min per wash.

Stage 4 - Antibody incubation

We’re ready to stain the protein loaded onto the membrane with antibodies. Detection is carried out with primary antibody and conjugated secondary antibody. In most protocols, both primary and secondary antibodies are used for detection, with primary and secondary playing crucial roles in target recognition and signal amplification. Optimizing antibody concentrations, including determining the optimal primary antibody concentration and the appropriate secondary concentration pair, is essential for achieving accurate and reliable results. It is also important to perform secondary antibody validation to ensure specificity and avoid non-specific signals. The use of primary and secondary antibodies is critical for signal amplification and specificity validation in protein detection assays.

Materials required

• Prepared membrane

• Primary antibody

• Secondary antibody (for example ab97051, HRP conjugated)

• Blocking buffer (for example 5 % non-fat dry milk (NFDM) or BSA in TBST)

• Wash buffer (TBST)

Steps

Dilute the primary antibody in the blocking buffer.

Incubate the membrane in the pre-diluted primary antibody prepared during the previous step.

• Add ~ 10–20 mL of the pre-diluted primary antibody to the membrane.

• Incubate for 1 h at room temperature on a shaker.

Wash the membrane three times with wash buffer.

• 10 min per wash with gentle shaking.

Incubate the membrane with a secondary antibody.

• 30 min at room temperature

Wash the membrane three times with TBST.

• 10 min per wash with gentle shaking.

Stage 5 - Detection

We’re now ready to visualize the targeted protein on the membrane using an ECL substrate. Including a positive control on the membrane is essential to validate the results and ensure the assay's accuracy. During detection, you can also confirm antibody specificity by verifying that the antibody binds selectively to the target protein. After visualization, quantitative analysis can be performed to measure and compare protein levels in the detected spots.

Materials required

• ECL detection reagent (for example ab133406)

Steps

Prepare the chemiluminescent substrate reagent as recommended by the manufacturer.

Place the membrane on the plastic board and incubate the blot with at least 500 μL ECL substrate reagent for 2–5 mins.

• Cover it with Saran wrap to remove the excess solution from the surface.
• Store the substrate at 4°Cand preheat it for 30 mins at RT before use.

Discard the substrate and soak up the remaining liquid with soft paper.

• Cover the plastic board with transparent plastic film.
• Keep the membrane wet.

Visualize the membrane for 1 sec–3 mins using any appropriate system that can take chemiluminescent images.

• Use the white-light picture to double-check the results