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WB Troubleshooting

No signals from target band? So many Non-specific bands? So dark is the image background? Various shapes of the bands? The methods to solve the problem may be here!


The western blot (sometimes called the protein immunoblot), or western blotting, is a widely used analytical technique in molecular biology and immunogenetics to detect specific proteins in a sample of tissue homogenate or extract.
Western blot has developed rapidly in recent years and has become a major technique for protein research, however, because of its complicated process and key points, it is time-consuming and each step may affect the final result.
There have been a number of web literature, which have already summarized and analyzed the common problem of WB experiments. This article will combine the experience of previous literature and give out the solution and suggestion.

Basic process of Western Blot

Firstly, let's review the experimental process of Western blot (as shown in the figure on the right). The western blot experiment is a complicated process that includes several steps and factors affecting the results, such as gel concentration of SDS-PAGE , transmembrane buffer, blocking solution, primary antibodies and their specificity, secondary antibodies incubation and rinsing.


Summary of common problems

This article summarizes the phenomenon into the following five categories, analyzes the causes of the problem, and provides a solution to the problem for references.

1. No signals or no target band on the membrane
2. Weak signals in the target band
3. Images so dark
4. So many non-specific bands
5. Abnormal shape of the band

I). No signals or no target band on the membrane

Image Image

II). Weak signals in the target band

Image Image

III). Images so dark







IV). So many non-specific bands

Image Image

V). Abnormal shape of the band



There may be two reasons for this phenomenon. One is the problem with the gumming configuration, which causes the glue inhomogeneous after solidification. We should reconfigurate the gumming and ensure the quality of gels. The second are the impurities inside the samples. We could centrifuge the samples before use to remove impurities as much as possible.

White shape inside the bands


The possible reason for this phenomenon is that the concentration of the primary antibody is too high, the HRP catalytic activity on the secondary antibody is too strong, and the chromogenic substrate is at the critical point, so that the reaction time is not long enough, and the surrounding substrates are completely catalyzed.The concentration of the primary and secondary antibodies can be reduced, or the substrate can be replaced.

Band tailing


The main reason for this phenomenon is that the volume of protein is too large, or the concentration and time of the primary antibody are too long. The volume of protein can be adjusted according to the situation, and the concentration and time of the primary antibody can also be shortened.

Bands are connected


This may be due to sample overloading or a gumming problem, or there are gaps between the separating and concentrating gels.  This problem can be avoided by reducing the sample volume and improving the quality of the gumming.

Smile-shaped bands


The shape of bands like ︶ appears. The reason is that the speed of electrophoresis is too fast, which could be slowed down by reducing the voltage; or the electrophoresis temperature is too high, which makes the gel deformed, we can put the electrophoresis into a cold room or in ice bath or change the pH value.

White circles inside the bands


This phenomenon is most likely due to the existence of bubbles between the film and gel during the electrical transfer, it is recommended to remove the bubbles between the film and gel before the transfer.


Western blot is currently one of the most commonly used methods for protein research, but its results are also unpredictable because of its complicated procedures. However, as long as the experimental procedure is standardized, suitable reagents and stable imaging system are used, we believe that everyone can produce "beautiful" bands.

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