Choosing Indirect vs Direct Fluorescence for Your Experiment

In research, antibodies are a very important tool. They tag the protein of interest with a specific antibody, meaning that flow cytometry (FACS), Western blot, ELISA, and fluorescence microscopy can be performed to study interactions, location, and more.

  • As it utilizes a single antibody against the protein of interest, direct immunofluorescence is streamlined. The primary antibody is directly conjugated to a fluorophore.
  • Indirect immunofluorescence uses two antibodies and is a multi-step process. The primary antibody is unconjugated but raised in a specific animal. Then the secondary antibody is specific to the primary antibody and is conjugated to a fluorophore that can be excited to show the protein of interest.

Overview of the main steps of direct and indirect IF.

Figure 1. Overview of the main steps of direct and indirect IF. More details here. Image Credit: Proteintech Group, Inc.

Recently, Proteintech launched a product line called CoraLite. It is a series of fluorescent-dye conjugated antibodies which can be employed in numerous applications, including immunofluorescence. CoraLite enables scientists to create beautiful IF images from direct labeling, as seen in Figure 2.

Immunofluorescent analysis of (4% PFA) fixed HepG2 cells using CL488-66037 (Green, ATP5A1 antibody) at dilution of 1:100 and CL594-66187 (Red, Cytokeratin 18 antibody) at dilution of 1:100.

Figure 2. Immunofluorescent analysis of (4% PFA) fixed HepG2 cells using CL488-66037 (Green, ATP5A1 antibody) at dilution of 1:100 and CL594-66187 (Red, Cytokeratin 18 antibody) at dilution of 1:100. Image Credit: Proteintech Group, Inc

Direct vs. indirect IF

Direct and indirect IF methods each have advantages and drawbacks, so considering the qualities of your protein of interest and the desired outcome of an experiment will help you to decide between the two methods. The figure below can be used to compare the two methods.

Image credit: Proteintech

What are the advantages of directly conjugated antibodies?

  • For multiplex imaging, directly conjugated antibodies are perfect for pairing with other antibodies. There is also no chance of cross-reaction with another primary antibody.
  • Directly conjugated antibodies avoid non-specific binding, which often happens with the secondary antibody step, enabling a clearer image to be gathered in immunofluorescence. There is also no chance of cross-reaction with another primary antibody.
  • Additionally, the experimental design is simplified as there is no need to carry out an extra step using a secondary antibody.

When the antigen being targeted is in high abundance, this is when directly conjugated antibodies work the best. This is because, unlike in indirect staining, where several secondary antibodies will bind to a single primary, the signal is not amplified with the two-step process.

It is worth considering that autofluorescence will still need to be checked for, and good positive control (IF tips) must be kept.

About Proteintech Group, Inc.

Proteintech are a global biotech company and a renowned center of excellence for the manufacture and supply of quality antibodies, ELISA kits and proteins to the life science research community.

With offices in the US (Chicago), UK (Manchester) and China (Wuhan) Proteintech are always available to support your research. Part of Proteintechs early vision was to make all its own products, to the highest standards possible and to take complete responsibility for the quality. With an emphasis on developing antibodies from whole proteins, Proteintech provides researchers with unmatched reliability and reproducibility.


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Last updated: Mar 23, 2020 at 7:28 AM

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