Common Questions and Solutions for WB

Protein blotting or immunoblotting (Western blot, WB) is a common laboratory method used to detect proteins and assess their expression levels. The protein of interest is identified based on its molecular weight and immunoreactivity with specific antibodies. Protein blotting consists of a series of interconnected steps (Figure 1).

Due to the long duration and intricate details of this experiment, from sample preparation to development, problems may arise at each step, resulting in bands that are often peculiar and unsatisfactory... In this article, we will introduce some common issues in WB experiments and their corresponding solutions^[2].

01. Smiling Bands

• Reason: Too fast migration, high temperature of the electrophoresis buffer, protein overload, or insufficient running buffer inside the well.

• Suggestion: Reduce migration speed, pre-cool the buffer, decrease the amount of protein loaded, ensure the buffer fully covers the well and that buffer in the inner chamber does not leak^[4][6].

02. Frowning Bands

• Reason: Improper equipment setup, which may be due to bubbles at the bottom of the gel and glass plates, or incomplete polymerization on both sides.

• Suggestion: This issue can be avoided by adjusting the setup.

03. Trailing Bands

• Reason: Poor sample dissolution; certain degree of protein degradation; repeated use of electrophoresis buffer.

• Suggestion: Load samples after they are fully dissolved and mixed; try to use fresh samples; use freshly prepared electrophoresis buffer.

04. Dumbbell-shaped Uneven Bands

• Reason: Issues with gel formulation, unevenness after the gel solidifies; sample may contain too many impurities.

• Suggestion: Reconfigure the gel and make sure there's no problem with its quality to avoid this phenomenon; centrifuge the sample before using.

05. Sticking Bands

• Reason: It could be due to excessive sample loading, or issues with gel preparation. There might be a gap between the separating gel and the stacking gel causing sample leakage.

• Suggestion: This can be avoided by reducing the sample loading amount and improving the quality of gel preparation.

06. Background Bubbles

• Reason: There are bubbles on the membrane during transfer.

• Suggestion: Ensure all bubbles between the gel and blotting membrane are removed when assembling the "sandwich".

07. Uneven Black Spots on the Background

• Reason: The blocking solution is not fully dissolved, causing insoluble particles to adhere to the membrane and form black spots when luminescence occurs; or uneven distribution of the antibody on the membrane.

• Suggestion: Ensure the blocking solution is fully dissolved. After blocking, wash three times with TBST before adding the primary antibody. Keep shaking during antibody incubation.

08. Blank Spots in Bands

• Reason: There's a blank spot in the middle of the target protein band, but the surrounding background is normal. This phenomenon may be due to excessively high concentrations of primary and secondary antibodies, which causes rapid substrate consumption, leading to the formation of blank spots when chemiluminescence is performed as the substrate is exhausted.

• Suggestion: You can reduce the amount of protein loaded; lower the concentration of primary and secondary antibodies; or dilute the developing solution.

Through this article, we believe that common problems are no longer a challenge for you! If the content above does not solve your problem, please feel free to leave a comment below. Little M welcomes everyone to actively discuss various challenging issues encountered in experiments~