FNDC5 Antibody, Biotin conjugated

What is FNDC5 and how does it relate to irisin?

FNDC5 is a transmembrane protein that undergoes proteolytic cleavage to release irisin, a 12 kDa peptide hormone. The full FNDC5 protein appears at approximately 20-25 kDa in Western blots, while the cleaved irisin peptide appears at approximately 12 kDa. Immunoblotting analyses using monoclonal antibodies against FNDC5 show different bands at various molecular weights, with specific bands at 25, 20, and 12 kDa representing irisin glycosylated isoforms, dimers, and/or complete FNDC5 protein . The FNDC5 gene encodes this protein, with expression observed in various tissues including muscle and adipose tissue, and the resulting irisin functions as a myokine/adipokine signaling molecule.

How is FNDC5/irisin produced physiologically?

FNDC5/irisin secretion appears responsive to physiological stimuli. Studies have observed that secretion of FNDC5/irisin by adipose depots increases with short-term exercise and reduces during fasting conditions . The protein follows a specific expression profile during adipocyte differentiation, being absent in pre-adipocyte secretomes but showing increasing presence from differentiation day 2 onward .

What are the primary applications for FNDC5 antibody, biotin conjugated?

The biotin-conjugated FNDC5 antibody (such as ABIN1169431) is primarily used for Western Blotting (WB) and ELISA applications . The biotin conjugation enhances detection sensitivity through avidin-biotin complex formation. This antibody can detect endogenous mouse and human irisin, making it valuable for studying native expression patterns. Additional applications might include immunofluorescence in cultured cells, though specific validation for each application is recommended.

What is the specificity profile of FNDC5 antibody, biotin conjugated?

The FNDC5 antibody shows reactivity against human, mouse, rat, and monkey FNDC5/irisin proteins . According to available documentation, the polyclonal antibody ABIN1169431 recognizes both the full FNDC5 protein and the secreted irisin peptide. The antibody has been raised against recombinant irisin, targeting amino acid sequences that allow for detection of both FNDC5 and cleaved irisin across multiple species .

What molecular weight bands should be expected when using FNDC5 antibody in Western blotting?

When performing Western blotting with FNDC5 antibody, researchers should expect to observe:

• Full FNDC5 protein at approximately 20-25 kDa

• Glycosylated isoforms at approximately 25 kDa

• Cleaved irisin peptide at approximately 12 kDa

• Potential dimers at higher molecular weights

Two-dimensional (2-DE) Western blotting has confirmed these observations, detecting exclusive spots in differentiated adipocytes with the expected molecular weight and isoelectric point for 12 kDa-secreted non-glycosylated irisin . The mouse FNDC5 is predicted at 20.3 kDa with a pI of 5.7, while mouse irisin is predicted at 12.6 kDa with a pI of 4.99 according to Uniprot .

How should samples be prepared for optimal FNDC5/irisin detection?

Based on experimental findings, both intracellular and secreted fractions can be analyzed for FNDC5/irisin. For secretome analysis, cell culture media from differentiated cells yields best results, as irisin secretion increases with differentiation . To reduce non-specific binding, particularly in the high molecular weight areas, inclusion of a blocking peptide is recommended when performing immunoblotting. Two-dimensional electrophoresis (2-DE) followed by Western blotting has proven effective for distinguishing specific FNDC5/irisin signals from non-specific antibody reactions .

What controls should be included when studying FNDC5/irisin expression?

Several controls are critical when studying FNDC5/irisin:

• Blocking peptide control: Antibody incubation with a specific blocking peptide can reveal unspecific binding, particularly in high molecular weight regions

• Commercial irisin peptide: Running commercial irisin peptide alongside samples helps confirm band identity

• FNDC5 knockout/knockdown samples: Samples with FNDC5 gene silencing provide the gold standard negative control, as demonstrated in studies where spots with expected isoelectric point and molecular weight for FNDC5/irisin disappear after FNDC5 gene silencing

• Cross-species validation: Loading murine and human samples together can confirm consistent detection of the 12 kDa irisin band across species

How can FNDC5/irisin detection be validated across different experimental systems?

Validation across experimental systems can be achieved through:

• Multi-technique confirmation: Combining immunoprecipitation, 2-DE Western blotting, and mass spectrometry

• Cross-species comparison: Human and mouse FNDC5/irisin are 96.698% identical (as shown in Supplementary Figure 1 referenced in ), facilitating cross-species validation

• Antibody blocking: Confirming specificity by demonstrating reduced signal when using blocking peptides

• Genetic manipulation: Using FNDC5 knockdown or knockout systems to confirm antibody specificity

What are the known limitations and controversies regarding FNDC5/irisin detection?

Several important limitations and controversies exist in the field:

• Antibody specificity concerns: Multiple studies have raised questions about the specificity of commercially available antibodies for FNDC5/irisin detection

• ELISA kit reliability: Researchers have debated whether ELISA kits for irisin provide accurate measurements, with Albrecht et al. (2015) specifically arguing that some ELISA kits may not be accurate

• Cross-reactivity issues: Non-specific binding in high molecular weight areas has been observed and requires careful control experiments

• Reproducibility challenges: Variations in preservation conditions, temperature, antibody lots, and operational contingency can influence detection results

How can non-specific binding be minimized when using FNDC5 antibody?

To minimize non-specific binding:

• Use blocking peptides to identify and eliminate non-specific signals

• Perform 2-DE Western blotting to separate proteins by both molecular weight and isoelectric point

• Include appropriate negative controls such as FNDC5 knockout/knockdown samples

• Optimize blocking conditions and antibody concentrations through titration experiments

• Verify signals using multiple detection methods and antibodies targeting different epitopes

What factors influence the variability in FNDC5/irisin detection across studies?

Several factors contribute to variability:

• Antibody heterogeneity: Different commercial antibodies target various epitopes and may have different specificities

• Sample preparation differences: Variations in protein extraction methods can affect detection

• ELISA kit limitations: As noted by researchers, ELISA results can be influenced by preservation conditions, temperature, antibody quality, and operational contingency

• Biological variables: Age, gender, race, disease duration, and physiological state (exercise, fasting) all influence FNDC5/irisin expression levels

• Technical variations: Differences in immunoprecipitation efficiency, gel electrophoresis conditions, and transfer methods

How does FNDC5/irisin expression relate to cognitive function and neurological disorders?

FNDC5/irisin has emerged as a potential factor in cognitive function and neurological disorders, particularly in elderly populations. Research indicates that FNDC5/irisin may have neuroprotective effects and could potentially influence cognitive decline and dementia progression. The exact mechanisms remain under investigation, but exercise-induced increases in FNDC5/irisin may contribute to cognitive benefits. Despite promising findings, researchers note that many studies remain at experimental stages, with limited direct studies of irisin on central autophagy and conflicting results regarding plasma irisin level alterations in patients with dementia .

What new detection methods might improve FNDC5/irisin measurement?

Given the limitations of current detection methods, several promising approaches deserve investigation:

• Advanced sensor technologies: Development of sensors with improved specificity for FNDC5/irisin detection

• Nanotechnology applications: Utilizing nanomaterials for enhanced detection sensitivity and specificity

• Mass spectrometry-based quantification: Implementing targeted proteomics approaches for absolute quantification

• Comparative validation studies: Conducting systematic comparisons of available ELISA kits and antibodies

• Aptamer-based detection systems: Developing aptamers with high binding affinity and specificity for FNDC5/irisin

What critical gaps remain in understanding FNDC5/irisin biology?

Despite progress, several critical knowledge gaps persist:

• Translation of findings from mice to humans: Some researchers have questioned whether beneficial effects observed in mice can be translated to humans

• Standardization of detection methods: The field requires more standardized approaches for measuring FNDC5/irisin levels

• Large-scale clinical validation: Large-scale clinical research and long-term follow-up studies are needed to clarify relationships between FNDC5/irisin and various health outcomes

• Mechanistic understanding: More research is needed to elucidate the precise mechanisms by which FNDC5/irisin influences processes like cognition, metabolism, and cancer progression

• Regulation of FNDC5/irisin production: Further investigation into the factors controlling FNDC5 expression and irisin release in different physiological states

What therapeutic potentials exist for targeting the FNDC5/irisin pathway?

Based on current research, several therapeutic directions warrant exploration:

• Exercise mimetics: Development of interventions that mimic exercise-induced FNDC5/irisin production

• Cognitive protection: Investigating FNDC5/irisin as a potential therapeutic target for dementia and cognitive decline

• Cancer treatment: Exploring the tumor-suppressive properties of FNDC5/irisin for cancer therapy, particularly in gastric cancer where low FNDC5 expression correlates with poor outcomes

• Metabolic regulation: Targeting FNDC5/irisin pathways for metabolic disorders

• Personalized exercise protocols: Optimizing exercise regimens based on individual FNDC5/irisin responses to maximize health benefits