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Choosing the right antibody for resistin-like molecule (RELM/FIZZ) family members
Chunling Fan1, Brian A Johns, Qingning Su
1Department of Anesthesiology and Critical Care Medicine, Johns Hopkins University School of Medicine, Baltimore, MD, 21205, USA.
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.
Insights
Antibody specificity is crucial for studying resistin-like molecules (RELM). This research validated RELM isoform antibodies, revealing issues with cross-reactivity and applicability in Western blots and immunofluorescence assays.
Area of Science:
- Biochemistry
- Immunology
- Cell Biology
Background:
- Resistin-like molecules (RELM), also known as found in inflammatory zone (FIZZ) proteins, play significant roles in physiology and pathophysiology, particularly in inflammation.
- RELM isoforms exhibit high sequence and structural similarity, possess distinct yet overlapping functions, necessitating specific detection methods.
- Existing research often focuses on single isoforms and lacks rigorous validation of antibody specificity, potentially leading to inaccurate interpretations.
Purpose of the Study:
- To establish and characterize HEK 293 cell lines expressing individual RELM isoforms.
- To determine the specificity of commercially available and custom-generated antibodies against RELM isoforms using Western blot and immunofluorescence.
- To highlight the importance of antibody specificity in RELM research.
Main Methods:
- Construction and establishment of HEK 293 cell lines stably expressing each mouse and human RELM isoform.
- Western blot analysis to assess antibody specificity and detect cross-reactivity.
- Immunofluorescence microscopy to evaluate antibody applicability and specificity in cellular contexts.
Main Results:
- Established stable HEK 293 cell lines for each RELM isoform.
- Identified antibodies with varying specificities; some specific in Western blots were not suitable for immunofluorescence.
- Observed cross-reactivity issues with certain antibodies in Western blot assays, indicating potential for misidentification of RELM isoforms.
- Demonstrated that antibody performance can differ between Western blot and immunofluorescence techniques.
Conclusions:
- Antibody specificity is a critical consideration for accurate research involving RELM isoforms.
- Validated cell lines provide a reliable tool for assessing antibody specificity.
- Researchers must carefully validate antibody performance across different techniques to ensure reliable data interpretation in RELM studies.
