Choosing the right antibody for resistin-like molecule (RELM/FIZZ) family members

Abstract

The family of resistin-like molecules (RELM), also known as found in inflammatory zone (FIZZ), consists of four members in mouse (RELMα/FIZZ1/HIMF, RELMβ/FIZZ2, Resistin/FIZZ3, and RELMγ/FIZZ4) and two members in human (resistin and RELMβ). The importance of these proteins in many aspects of physiology and pathophysiology, especially inflammatory processes, is rapidly evolving in the literature, and many investigators are beginning to work in this field. Most published studies focus on only one isoform, do not evaluate other isoforms that might be present, and have not tested for the specificity of the antibody used. Because RELM isoforms have high sequence and structural similarity and both distinct and overlapping functions, it is important to use a specific antibody to distinguish each isoform in the study. We constructed and established HEK 293 cell lines that constitutively express each isoform. Using these cell lines, we determined the specificity of antibodies (both commercially available and laboratory-made) to each isoform by Western blot and immunofluorescence. Some of the antibodies showed specificity in Western blotting but were not applicable in immunofluorescence. Others showed cross reactivity in Western blot assays. Our results indicate that RELM antibody specificity should be taken into account when using them in research and interpreting data obtained with them.

Fig. 1

Specificity of mRELMα antibodies in Western blot and immunofluorescence assays. Antibodies from R&D Systems (A) and our laboratory (B) specifically recognized their target protein on Western blots. When used for immunofluorescence, goat anti-mRELMα antibody from R&D Systems bound only to mRELMα protein in HEK 293 cells (C). Mouse anti-FLAG antibody from Sigma was used to indicate the inductive expression of fusion protein in HEK 293 cells. Cy5-conjugated donkey anti-goat IgG was used as secondary antibody (C,purple).

Fig. 2

Specificity of mRELMβ antibodies in Western blot and immunofluorescence assays. Although antibodies from Peprotech (A) and Affinity Bioreagents (ABR) recognized mRELMβ on Western blots, they exhibited weak cross-reactivity to mRELMγ. Rabbit anti-mRELMβ from Dr. Gary Wu’s laboratory (University of Pennsylvania) exhibited strong specificity for mRELMβ (C). (D) When used for immunofluorescence, rabbit anti-mRELMβ antibody from ABR recognized mRELMβ and mRELMγ equally. (E) Rabbit anti-mRELMβ from Dr. Wu’s lab exhibited good recognition of its target protein mRELMβ after cells were treated with SDS to enhance antigenicity. Mouse anti-FLAG antibody from Sigma was used to indicate the inductive expression of fusion protein in HEK 293 cells. Cy5-conjugated donkey anti-rabbit IgG (D, purple) and Cy2-conjugated donkey anti-rabbit IgG (E, green) were used as secondary antibodies.

Fig. 3

Specificity of mResistin antibody in Western blot and immunofluorescence assays. Rabbit anti-mResistin from LifeSpan BioSciences (LSBio) specifically recognized mResistin when used for Western blotting (A) and immunofluorescence (B). Mouse anti-FLAG antibody from Sigma was used to indicate the inductive expression of fusion protein in HEK 293 cells. Cy3-conjugated donkey anti-rabbit IgG (C, red) was used as secondary antibody.

Fig. 4

Specificity of mRELMγ antibodies in Western blot and immunofluorescence assays. (A) Rabbit anti-mRELMγ antibody from Alpha Diagnostics Inc. (ADI) recognized both mRELMα and mRELMγ. (B and C) The two anti-mRELMγ antibodies produced in our laboratory against two different peptide antigens showed fair specificity for mRELMγ on Western blot, although one (#1, B) exhibited weak binding to mRELMβ. However, in the immunofluorescence assay, antibody #1 bound all mRELM isoforms (D). After cells were treated with SDS, antibody #2 that had initially shown no binding, exhibited binding only to mRELMβ protein (4E). Mouse anti-FLAG antibody from Sigma was used to indicate the inductive expression of fusion protein in HEK 293 cells. Cy5-conjugated donkey anti-rabbit IgG (D, purple) and Cy2-conjugated donkey anti-rabbit IgG (E, green) were used as secondary antibodies.

Fig. 5

Specificity of hResistin antibodies in Western blot and immunofluorescence assays. Goat anti-hResistin from R&D Systems specifically recognized hResistin on Western blots (A), whereas rabbit anti-hResistin from Santa Cruz Biotechnology showed a preference for binding to mRELMβ and only weak binding to hResistin (B). (C) The anti-hResistin antibody from R&D Systems also exhibited specificity for hResistin in the immunofluorescence assay. Mouse anti-FLAG antibody from Sigma was used to indicate the inductive expression of fusion protein in HEK 293 cells. Cy5-conjugated donkey anti-goat IgG was used as secondary antibody (C, purple).

Fig. 6

Specificity of hRELMβ antibodies in Western blot and immunofluorescence assays. In Western blot assays, hRELMβ antibodies from Acris (A), Adipogen, and Chemicon (B) were each highly specific for hRELMβ when tested against all RELM isoforms, including mouse. (C) Only the goat anti-hRELMβ antibody from Acris exhibited binding to hRELMβ in the immunofluorescence assay. Mouse anti-FLAG antibody from Sigma was used to indicate the inductive expression of fusion protein in HEK 293 cells. Cy2-conjugated donkey anti-goat IgG was used as secondary antibody (C, green)