Positive and Negative Controls

You should include both positive and negative controls in your experimental design to serve as helpful checkpoints for accurate target detection.

Positive Control

Positive controls are essential for establishing experimental validity. A positive control ensures that the antibody recognizes your target protein within the experimental conditions.

Examples of positive controls are lysates overexpressing the gene encoding your protein of interest or a pure sample of your protein of interest. You could develop positive controls in your lab. Alternatively, many companies sell them along with an antibody validated with that control.

A signal in the lane loaded with positive control indicates that the antibody is specific to the protein of interest and all steps in the protocol worked correctly1. Absence of signal for a positive control could mean that the antibody may not be specific to the antigenic epitopes or the blotting protocol affected the target protein1.

Negative Controls

Negative controls will help you rule out any non-specific binding of antibodies to components in your sample. A negative control helps validate that the signal you see is from the binding of the antibody to the target antigen epitope only, and not from background interaction.

An example of an appropriate negative control could be loading buffer without sample or lysate that is known to not contain your target of interest2,3.

Presence of signal in the lane loaded with a negative control indicates that antibodies may be interacting with non-specific targets or components within sample/loading buffers.

Blot with positive and negative control lanes
Incorporate suitable control samples in your study design to validate the results of the experiment. Positive and negative controls provide helpful checkpoints to confirm specificity of the antibody to target.
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References

  1. Biocompare Bench Tips: Improve Western Blots by Including a Positive Control. Posted Aug 7, 2013.
  2. Western blotting guide and troubleshooting. MD Biosciences.
  3. European Monoclonal Antibodies Network (Euro MabNet). Antibody Validation: Practical Guide to Finding and Validating Suitable Antibodies for Research.

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