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Dot Blot-Protocol & Troubleshooting

Dot blot is a simple and rapid technique for the detection of proteins. It is similar to western blotting, but does not require separation by electrophoresis. It is a procedure in which the sample is applied directly to a spot on the membrane and then the blotting procedure is performed. This saves significant time but does not provide information on the size of the target protein.

If your goal is to simply detect the presence of the target protein and you have purified samples of the target protein and specific antibodies. Then you can use dot blot as the technique of choice. Creative Biolabs provides a good general protocol. As well as on this page we share with you some experimental troubleshooting tips on dot blot.

For a quick and comprehensive selection of antibodies, check out our product list.

Solutions and Reagents

Stages Solutions and Reagents
Membrane Preparation Blocking buffer, dilution buffer, membrane washing buffer
Blotting Primary antibody, secondary antibody, antibody dilution buffer, membrane washing buffer, substrate

Dot Blot Procedure

Dot blot is a simplified method that is also based on the principle of antibody recognition and binding to antigen. It can be used to determine the presence of a target protein in a sample or whether an antibody is effective. The following protocol is a good general method for each researcher to build their own experimental protocol.

Dot Blot

1. Membrane Preparation

Cut a suitable piece of nitrocellulose membrane according to the sample size. Mark the membrane so that you can track the blotted samples. Secure the blotting membrane to prevent curling. Slowly aspirate the prepared sample onto the membrane at a defined location. Allow the membrane to dry.

2. Blocking

Select the appropriate blocking buffer. Use an excess amount of blocking buffer to completely cover the membrane. Shake for blocking at room temperature. Decant the blocking solution and rinse the membrane several times with wash buffer.

3. Antibody Incubation

Dilute the antibody with dilution buffer. Cover the membrane with sufficient diluted primary antibody solution and incubate on a rotary shaker. Discard the diluted primary antibody and wash several times with wash buffer. After washing, incubate the membrane with diluted secondary antibody. After incubation is complete, discard the diluted antibody solution and repeat the wash.

4. Detection and Imaging

Select the appropriate substrate for incubation. Pick up the blotting membrane to drain excess reagent for chemiluminescence/fluorescence detection.


In this section, we will discuss the problems that can arise when performing dot blot techniques. In particular, how to avoid low signal, high background and non-specific binding.

Weak signal

  1. Sample causes. One reason for seeing a lack of signal could be a lack of protein. This may come from improper sample preparation. When homogenizing samples, be sure to do so on ice, as well as using an appropriate ice-cold buffer. If you know that your protein is located in a specific compartment, be sure to use lysis conditions that enrich the protein in that compartment.
  2. Blocking causes. The initial blocking of the membrane is very important. Excessive blockage can lead to poor binding and problems in detecting antigens. We typically block for 1 hour. Or you can use a proprietary blocking solution.
  3. Antibody causes. If an unsuitable antibody is used, either primary or secondary antibody, the signals will not show up. Low concentrations of antibodies may result in weak signals, and increase the exposure time to help make clearer.
  4. Incubation causes. When incubating antibodies, BSA or skim milk is typically used. This may mask the antigen, so should be used in reduced amounts when this occurs.
  5. Reagent and solution causes. Check the quality of the reagents and solutions used and make sure that the buffer solution is not contaminated.
  6. Washing causes. Long wash times can also lead to weakening of the signal. We recommend that you take the correct washing procedure.

High background

  1. Antibody concentration causes. Due to the high antibody concentration, the antibody will bind to the PVDF membrane. We recommend that you preferably serial dilution of your samples and antibodies. This can help you determine the optimal antibody and sample concentrations for dot blot and reduce background.
  2. Blocking causes. The initial blocking of the membrane is very important. Insufficient closure (too short a time in the closure buffer) can lead to non-specific binding of primary and secondary antibodies and result in very high background.
  3. Washing causes. A high background may be due to the presence of excess antibodies still present. To ensure that the signal you observe is due to the binding of your target antigen and antibody and not excess antibody, you can add a proper washing step as appropriate.

Dot blot is basically a simplified approach to western blotting, so the troubleshooting that applies to western blotting also applies to dot blot. The time you can spend trying various conditions can help you avoid some headaches. In addition, it can save you the trouble of repeating the experiment unnecessarily.

We provide a general workflow for the simple capture of proteins onto membranes by dot blotting techniques. The method described is a simple and fast process. We hope that we have provided relevant support and resources to help you understand the dot blotting technique.

Antibodies play a crucial role in this technique, so you can check our related products and choose the right antibody for your experiment.


  1. Bonin S and Dotti I. General Protocol for Dot-Blot. In: Guidelines for Molecular Analysis in Archive Tissues. Springer, 2011.

For research use only. Not intended for any clinical use.

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