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