mc4r Antibody

What is MC4R and why is it an important research target?

MC4R (melanocortin 4 receptor) is a G-protein coupled receptor encoded by the MC4R gene in humans. It functions as a key switch in the leptin-melanocortin molecular axis controlling hunger and satiety. This 332 amino acid protein (36.9 kDa) is also known as BMIQ20, melanocortin receptor 4, and MC4-R . MC4R has become an important research target because of its central role in energy homeostasis and weight regulation. Understanding its signaling pathways, activation mechanisms, and variants has implications for metabolic disorders, obesity research, and potential therapeutic development .

What species variants of MC4R are antibodies available for?

MC4R antibodies are available for multiple species, which is important for comparative studies and animal model research. Most commonly available antibodies target human MC4R, but antibodies reactive to other species variants are also produced, including those for canine, porcine, monkey, mouse, and rat MC4R . When selecting an antibody for cross-species research, it's essential to verify the species reactivity information provided by the manufacturer and consider sequence homology between species at the target epitope .

What are the common applications for MC4R antibodies?

MC4R antibodies are utilized in numerous research applications:

• Western Blotting (WB): For detection and quantification of MC4R protein in tissue/cell lysates

• Enzyme-Linked Immunosorbent Assay (ELISA): For quantitative measurement of MC4R

• Immunohistochemistry (IHC): For visualizing MC4R distribution in tissue sections

• Immunofluorescence (IF): For cellular localization studies

• Immunocytochemistry (ICC): For detection in cultured cells

• Flow Cytometry (FCM): For cell surface expression analysis

Each application requires specific antibody characteristics and validation. For example, antibodies used for Western blotting should be validated for specificity by showing appropriate molecular weight bands, while those for immunohistochemistry need to demonstrate specific tissue staining patterns consistent with known MC4R expression.

How do I verify MC4R antibody specificity?

Verifying antibody specificity is crucial for reliable research outcomes. For MC4R antibodies, consider these validation strategies:

• Testing on cells transfected with FLAG-tagged MC4R constructs versus untransfected controls

• Using ELISA to assess cell surface localization with anti-FLAG (M2) antibody detection

• Performing blocking experiments with the immunizing peptide

• Testing reactivity in MC4R knockout tissues/cells (negative control)

• Checking for cross-reactivity with other melanocortin receptor family members

• Verifying expected molecular weight (approximately 37 kDa) in Western blots

Researchers should also review published literature where specific antibodies have been previously validated, as this can provide valuable information on performance and reliability across different experimental conditions.

How can I assess MC4R cell surface expression in my experimental system?

Cell surface expression of MC4R is critical for proper receptor function and can be altered by mutations or experimental conditions. To assess MC4R cell surface localization:

• Use ELISA with epitope-tagged MC4R (e.g., FLAG tag) and surface-specific antibody detection:

  • Transfect cells with FLAG-tagged MC4R constructs

  • Fix cells with 3.7% paraformaldehyde (15 min, room temperature)

  • Block with 3% non-fat dry milk in 50 mM Tris-PBS pH 7.4

  • Incubate with anti-FLAG (M2) antibody (1:1000 dilution, overnight at 4°C)

  • Detect with HRP-conjugated secondary antibody (e.g., goat anti-mouse IgG)

• Alternative approaches include:

  • Flow cytometry with non-permeabilized cells

  • Confocal microscopy using immunofluorescence to distinguish membrane from intracellular staining

  • Biotinylation of cell surface proteins followed by streptavidin pulldown and Western blotting

The selection of method depends on the specific research question, available equipment, and required sensitivity.

How do MC4R antibodies differ from other experimental tools for studying MC4R function?

MC4R antibodies offer distinct advantages and limitations compared to other research tools:

Understanding these differences helps researchers select the most appropriate tools for specific experimental questions about MC4R biology.

How can I use antibodies to investigate MC4R variant functional consequences?

MC4R variants/mutations can have significant implications for receptor function. To investigate their consequences using antibodies:

• Compare cell surface expression levels between wild-type and variant MC4R:

  • Transfect cells with wild-type and variant MC4R constructs

  • Use cell surface ELISA or flow cytometry with MC4R antibodies

  • Quantify differences in surface expression

• Assess intracellular retention/misfolding:

  • Perform dual immunofluorescence with surface (non-permeabilized) and total (permeabilized) staining

  • Calculate surface-to-total ratio as an indicator of trafficking efficiency

• Combine with functional assays to correlate expression with activity:

  • Use cAMP accumulation assays (e.g., Lance Ultra HTRF) to measure signaling capacity

  • Monitor G-protein recruitment using specialized tools like ConfoSensor assays

This approach allows for semi-quantitative classification of variants as complete loss-of-function, partial loss-of-function, or wild-type-like, providing insights into the molecular mechanisms underlying MC4R-associated disorders.

What strategies can resolve contradictory results when using different MC4R antibodies?

Researchers often encounter conflicting results when using different MC4R antibodies. To resolve these contradictions:

• Compare epitope specificity:

  • Antibodies targeting different domains of MC4R (N-terminal, C-terminal, extracellular loops) may show different staining patterns

  • Map the precise epitope of each antibody (e.g., AA 1-50, AA 6-20, AA 25-57, cytoplasmic domain)

• Evaluate antibody validation evidence:

  • Review published literature for each antibody

  • Check manufacturer's validation data

  • Perform your own validation experiments (knockout controls, peptide competition)

• Implement multiple detection methods:

  • Use at least two antibodies targeting different epitopes

  • Complement antibody-based detection with other approaches (mRNA expression, reporter systems)

  • Confirm with functional assays when possible

• Consider protein conformation and post-translational modifications:

  • Some antibodies may preferentially recognize specific conformational states

  • Post-translational modifications may mask epitopes in certain cellular contexts

This systematic approach helps identify reliable antibodies and explains discrepancies between different experimental results.

How do I integrate MC4R antibody data with functional signaling assays?

For comprehensive MC4R characterization, integrating structural data from antibody studies with functional assessments is essential:

• Correlation of surface expression with signaling capacity:

  • Measure surface expression using antibody-based methods (ELISA, flow cytometry)

  • In parallel, assess cAMP accumulation using techniques like Lance Ultra HTRF

  • Calculate efficiency ratio (signaling output relative to surface expression)

• Monitoring conformational changes:

  • Use conformation-sensitive antibodies that preferentially bind active or inactive states

  • Complement with functional readouts like G-protein mimetic recruitment (ConfoSensor assay)

  • Advanced systems may use nanobodies that stabilize specific receptor conformations

• Data integration approach:

  • Plot surface expression versus functional activity for different MC4R variants

  • Classify variants based on combined datasets (trafficking defects, signaling defects, or both)

  • Use statistical approaches to normalize data across experimental systems

This integrated approach provides mechanistic insights into how structural alterations (detected by antibodies) translate to functional consequences in MC4R signaling pathways.