Western Blot: Indirect vs. Direct Detection

Overview

Antibody detection of the target protein is accomplished using a one-step or two-step protocol. The one-step procedure, direct detection, relies upon a single antibody which has been covalently joined to an easily detected label molecule (biotin, an enzyme, or a fluorescent dye). Labeled primary antibodies can be ordered directly from Bio-Rad or other commercial antibody suppliers. In addition, it is possible to directly label an antibody by using a commercially supplied labeling kit such as our LYNX Rapid Conjugation Kits®, or with in-house reagents.

With indirect detection, two different antibodies are used in sequence for the detection step. First, the Western blot is incubated with an unlabeled primary antibody directed against the target protein. After washing, a labeled secondary antibody is used to detect the presence of the primary antibody, and thus the target protein. The labeled secondary antibody is typically directed against the immunoglobulin class or subclass of the primary antibody’s species. For example, one of our popular secondaries is STAr88P, a purified donkey antibody raised against goat/sheep immunoglobulin (IgG) which is coupled to an HrP label. Biotinylated primary antibodies also require a two-step detection procedure; however, the second step involves incubation with streptavidin, a bacterial protein, conjugated to HrP (or AP), rather than with a labeled antibody.

Figure 11: Direct vs. Indirect Detection.

Comparison of Direct and Indirect Detection Methods

Direct Detection Indirect Detection
Advantages Advantages
  • Faster overall, since there are fewer steps.
  • Less chance of non-specific signal.
  • Often gives a stronger signal because multiple secondary antibodies bind to each primary antibody.
  • Easy to change label type or detection methods for a new experiment by swapping secondaries.
  • Saves labeling time and expense, especially when all primary antibodies are made in the same species.
  • Provides access to a wider range of labels.
Disadvantages Disadvantages
  • Coupling of label to the primary antibody may affect the antibody’s ability to bind to the target protein.
  • Labeling every primary antibody adds time and cost.
  • More non-specific signal can arise from the binding of the secondary antibody to other proteins on the blot.
  • Extra incubation and wash steps add time to the experiment.
Immunodetection: Blocking and Antibody Incubation Detection with Substrate