MC4R Antibody, HRP conjugated

What are the primary applications for MC4R Antibody, HRP conjugated?

MC4R Antibody, HRP conjugated is primarily utilized in Enzyme-Linked Immunosorbent Assay (ELISA) and immunohistochemistry on frozen sections (IHC-F) . The HRP conjugation eliminates the need for secondary antibody incubation, streamlining experimental workflows and potentially reducing background signal. The recommended dilution ranges are 1:500-1000 for ELISA applications and 1:100-500 for IHC-F . This reagent is particularly valuable for detecting MC4R expression patterns in brain tissue, where this receptor is predominantly expressed.

What species reactivity can be expected with commercially available MC4R Antibody, HRP conjugated?

The MC4R Antibody, HRP conjugated products available typically demonstrate reactivity with human, mouse, and rat samples . This cross-species reactivity is advantageous for comparative studies and translational research. When working with samples from other species, validation experiments should be conducted to confirm binding specificity, as sequence homology and epitope conservation may vary across species.

How should MC4R Antibody, HRP conjugated be stored to maintain optimal activity?

For optimal preservation of activity, MC4R Antibody, HRP conjugated should be stored at -20°C . The antibody is typically provided in an aqueous buffered solution containing 0.01M TBS (pH 7.4) with 1% BSA, 0.03% Proclin300, and 50% Glycerol . To minimize activity loss from repeated freeze-thaw cycles, it is strongly recommended to aliquot the antibody into multiple smaller volumes before freezing . When handling, keep the antibody on ice and return to storage promptly after use to prevent degradation of the HRP enzyme.

What protocol modifications are needed when using MC4R Antibody, HRP conjugated for Western blotting?

When using MC4R Antibody, HRP conjugated for Western blotting, several protocol modifications optimize detection sensitivity:

• Sample preparation: MC4R is a membrane-bound G protein-coupled receptor, requiring careful extraction protocols. Brain tissue samples should be lysed in ice-cold radioimmunoprecipitation assay (RIPA) buffer containing protease inhibitors .

• Protein loading: Load 30-50 μg total protein per lane for optimal detection .

• Blocking: Use 5% non-fat dry milk or 3% BSA in TBS-T for blocking membranes.

• Primary antibody incubation: Since the antibody is HRP-conjugated, dilute to 1:500-1:1000 in blocking buffer and incubate overnight at 4°C.

• Washing: Perform stringent washing (4-5 times with TBS-T) to remove unbound antibody.

• Detection: Proceed directly to enhanced chemiluminescence (ECL) detection without secondary antibody incubation.

• Controls: Include positive controls (tissue with known MC4R expression) and negative controls (MC4R knockout samples if available).

This protocol modification accounts for the direct detection capability of the HRP-conjugated antibody while addressing the technical challenges of detecting a seven-transmembrane receptor.

How can I validate the specificity of MC4R Antibody, HRP conjugated in my experiments?

Validating antibody specificity is crucial for reliable MC4R detection. A comprehensive validation approach includes:

• Positive and negative controls:

  • Test against samples with known MC4R expression levels (e.g., hypothalamic tissue vs. non-expressing tissues)

  • If available, use MC4R knockout samples as negative controls

• Peptide competition assay:

  • Pre-incubate the antibody with excess immunizing peptide (derived from human MC4R regions 1-50/332)

  • Compare signal with and without peptide competition

• Orthogonal methods:

  • Confirm expression patterns using alternative detection methods like RT-PCR

  • Compare results with other validated MC4R antibodies

• Signal localization:

  • Verify that detected signals correspond to expected subcellular localization (cell membrane)

  • Check for non-specific nuclear or cytoplasmic staining

• Cross-reactivity assessment:

  • Test against other melanocortin receptor subtypes (MC1R, MC2R, MC3R, MC5R) to confirm specificity

This multi-faceted validation approach ensures that experimental findings accurately reflect MC4R biology rather than non-specific interactions.

How can MC4R Antibody, HRP conjugated be used to study receptor trafficking in obesity-related MC4R variants?

MC4R Antibody, HRP conjugated provides valuable insights into receptor trafficking abnormalities associated with obesity-causing MC4R variants. A comprehensive experimental approach includes:

• Cell surface biotinylation assay:

  • Transfect cells with wild-type or mutant MC4R constructs

  • Use cell-impermeable biotinylation reagents to label surface proteins

  • Isolate biotinylated proteins with streptavidin and detect MC4R using the HRP-conjugated antibody

  • Compare surface expression levels between wild-type and mutant receptors

• Stimulation response analysis:

  • Treat transfected cells with α-MSH (100 nM, 3 hours)

  • Analyze membrane localization before and after stimulation

  • Quantify surface expression changes using immunofluorescence or surface biotinylation

• Confocal microscopy with dual labeling:

  • Use MC4R Antibody along with markers for intracellular compartments (ER, Golgi)

  • Determine where mutant receptors are retained within the cell

• Western blot quantification:

  • Compare total MC4R expression (whole cell lysates) versus membrane fraction

  • Calculate the surface/total ratio as a measure of trafficking efficiency

Research has demonstrated that obesity-associated MC4R variants (e.g., S85G, Y268H) show impaired membrane localization despite normal total protein expression, suggesting intracellular retention as a key pathogenic mechanism .

What methods can be used to assess functional consequences of MC4R variants using MC4R Antibody, HRP conjugated?

MC4R variants can lead to obesity through multiple mechanisms. A comprehensive functional assessment protocol leveraging MC4R Antibody, HRP conjugated includes:

• Signal transduction analysis:

  • Transfect cells with wild-type or mutant MC4R

  • Stimulate with α-MSH and measure downstream signaling

  • Use Western blotting with the following antibodies:

    • Phospho-PKA

    • Phospho-ERK1/2

    • Phospho-CREB

  • Quantify phosphorylation levels relative to total protein

• cAMP accumulation assay:

  • Perform HTRF or GloSensor® assays to measure cAMP production

  • Generate dose-response curves for α-MSH stimulation

  • Compare EC50 values between wild-type and mutant receptors

• Protein-protein interaction assessment:

  • Immunoprecipitate MC4R using the HRP-conjugated antibody

  • Identify binding partners by mass spectrometry

  • Compare interaction profiles between wild-type and mutant receptors

• Comparison with nanobody activation:

  • Test responsiveness to MC4R-specific nanobody agonists (e.g., pN162)

  • Determine if variants affect nanobody binding versus α-MSH binding

These methods allow researchers to determine if MC4R variants affect ligand binding, receptor activation, membrane localization, or downstream signaling, providing mechanistic insights into obesity pathogenesis.

What are common issues when using MC4R Antibody, HRP conjugated and how can they be resolved?

Working with MC4R Antibody, HRP conjugated can present several technical challenges. Here are common issues and their solutions:

• High background in immunohistochemistry:

  • Increase blocking time (2-3 hours with 5% BSA)

  • Use additional blocking agents (10% normal serum from host species)

  • Reduce antibody concentration (try 1:200-1:500)

  • Include 0.1% Triton X-100 in washing steps to remove non-specific binding

• Weak or absent signal:

  • Check tissue fixation (over-fixation can mask epitopes)

  • Perform antigen retrieval (citrate buffer pH 6.0, 95°C for 20 minutes)

  • Increase antibody concentration (1:50-1:100)

  • Extend incubation time (overnight at 4°C)

  • Ensure HRP activity is preserved (avoid repeated freeze-thaw cycles)

• Non-specific bands in Western blot:

  • Optimize blocking conditions (5% non-fat dry milk vs. 3% BSA)

  • Increase washing stringency (0.1% SDS in TBST)

  • Pre-adsorb antibody with non-specific proteins

  • Use gradient gels to better resolve proteins of similar molecular weight

• Inconsistent results:

  • Aliquot antibody to prevent freeze-thaw damage

  • Standardize sample preparation protocols

  • Include positive controls in each experiment

  • Maintain consistent incubation times and temperatures

These troubleshooting approaches address the specific challenges associated with detecting a membrane-bound G protein-coupled receptor while leveraging the advantages of HRP conjugation.

How can MC4R Antibody, HRP conjugated be optimized for dual labeling experiments?

Dual labeling experiments provide valuable insights into MC4R colocalization with other proteins. Optimization strategies include:

• Sequential detection approach:

  • Complete MC4R detection with HRP-conjugated antibody first

  • Quench HRP activity thoroughly (3% H₂O₂, 10 minutes)

  • Block again before applying the second primary antibody

  • Use alkaline phosphatase (AP) conjugated secondary antibody with different substrate

• Tyramide signal amplification (TSA):

  • Use MC4R Antibody, HRP conjugated at low concentration (1:1000)

  • Apply fluorophore-conjugated tyramide substrate

  • HRP catalyzes tyramide deposition, creating covalent binding to nearby proteins

  • Inactivate HRP completely before second antibody application

  • This approach provides signal amplification and permanent labeling

• Spectral separation optimization:

  • For DAB (brown) visualization of HRP, pair with:

    • Vector VIP (purple)

    • Vector SG (blue-gray)

    • AEC (red)

  • Choose complementary colors for clear visual distinction

• Controls for specificity:

  • Single-labeling controls to verify staining patterns

  • Species-specific secondary antibody controls

  • Absorption controls with immunizing peptides

This approach enables researchers to study MC4R colocalization with signaling partners, trafficking machinery, or other receptors with minimal cross-reactivity.

How can MC4R Antibody, HRP conjugated be used to investigate the role of MC4R in synaptic plasticity?

MC4R has emerging roles in synaptic plasticity beyond its established function in energy homeostasis. An experimental approach to investigate this using MC4R Antibody, HRP conjugated includes:

• Immunohistochemical analysis of receptor distribution:

  • Perform dual-labeling with MC4R Antibody and markers for:

    • Excitatory synapses (PSD-95, vGlut1)

    • Inhibitory synapses (Gephyrin, vGAT)

    • Specific neuronal populations (TH, ChAT, 5-HT)

  • Map MC4R distribution relative to synaptic structures

• Biochemical analysis of postsynaptic complexes:

  • Prepare synaptosomal fractions from relevant brain regions

  • Immunoprecipitate MC4R using the antibody

  • Analyze co-precipitated proteins by mass spectrometry

  • Focus on glutamate receptor subunits and scaffolding proteins

• Analysis of activity-dependent regulation:

  • Compare MC4R levels in synaptic fractions across conditions:

    • Basal state

    • After pharmacological stimulation (α-MSH)

    • After physiological challenges (fasting/feeding)

  • Quantify changes in synaptic localization

• Correlation with glutamate receptor subunits:

  • Analyze potential interactions between MC4R and glutamate receptor subunits

  • Western blot analysis of:

    • GluA1, GluA2, GluA3, GluA4

    • Phosphorylated forms (pS845-GluA1, pS831-GluA1, pS880-GluA2)

    • NMDAR subunits (NMDAR1, NMDAR2A, NMDAR2B)

Research has shown that MC4R deficiency causes dysregulation of postsynaptic excitatory neurotransmission , suggesting important interactions between melanocortin signaling and glutamatergic transmission in regions like the paraventricular hypothalamus.

What approaches can be used to study MC4R internalization dynamics using MC4R Antibody, HRP conjugated?

Understanding MC4R internalization dynamics is crucial for comprehending receptor desensitization and resensitization. A methodological approach using MC4R Antibody, HRP conjugated includes:

• Time-course analysis of receptor internalization:

  • Label surface MC4R with cleavable biotin

  • Stimulate with α-MSH for various durations (5, 15, 30, 60 minutes)

  • Cleave remaining surface biotin

  • Quantify internalized (protected) biotin-labeled MC4R using the HRP-conjugated antibody

  • Plot internalization rate curves

• Recycling assay:

  • Label surface MC4R with biotin

  • Induce internalization with α-MSH

  • Allow varying recovery periods

  • Quantify receptor recycling to the plasma membrane

  • Compare recycling kinetics between wild-type and mutant receptors

• Colocalization with endocytic markers:

  • Dual-label cells with MC4R Antibody and markers for:

    • Early endosomes (EEA1)

    • Recycling endosomes (Rab11)

    • Late endosomes/lysosomes (LAMP1)

  • Track receptor trafficking through the endocytic pathway

  • Determine the fate of internalized receptors

• Effect of β-arrestin recruitment:

  • Analyze β-arrestin binding to MC4R following stimulation

  • Compare wild-type and mutant receptors for differences in arrestin recruitment

  • Correlate with internalization rates

This methodological approach provides insights into how receptor dynamics influence MC4R signaling duration and intensity, which may be altered in pathological conditions involving the melanocortin system.

How can MC4R Antibody, HRP conjugated be used to evaluate novel MC4R-targeted therapeutics?

MC4R is a promising target for obesity treatment, and MC4R Antibody, HRP conjugated can facilitate therapeutic development through:

• Target engagement assessment:

  • Develop competitive binding assays between therapeutic candidates and the antibody

  • Determine if candidates and antibody recognize overlapping epitopes

  • Map binding domains through competition analysis

• Conformational change detection:

  • Compare antibody binding patterns before and after treatment with:

    • Peptide agonists (α-MSH analogs)

    • Small molecule modulators

    • Therapeutic nanobodies

  • Identify differential epitope accessibility reflecting conformational changes

• Receptor trafficking analysis:

  • Monitor how therapeutics affect:

    • Surface expression levels

    • Internalization rates

    • Recycling efficiency

  • Compare effects between wild-type and mutant receptors

• Downstream signaling assessment:

  • Analyze how therapeutics influence MC4R coupling to:

    • G protein-dependent pathways (cAMP/PKA)

    • G protein-independent pathways (β-arrestin/ERK)

  • Compare signaling profiles of different therapeutic modalities

Recent approaches using nanobodies targeting MC4R have shown promise, with agents like pN162 demonstrating potent full agonist activity (EC₅₀ ≈ 0.7 nM) and specificity for MC4R over other melanocortin receptor subtypes .

What methods can be used to study MC4R heterodimer formation using MC4R Antibody, HRP conjugated?

GPCRs like MC4R can form functional heterodimers with other receptors, potentially modifying signaling properties. Approaches to study MC4R heterodimers include:

• Co-immunoprecipitation analysis:

  • Immunoprecipitate MC4R using the HRP-conjugated antibody

  • Probe for co-precipitated receptors of interest

  • Compare heterodimer formation in different tissues/conditions

  • Include appropriate controls for non-specific binding

• Proximity ligation assay (PLA):

  • Use MC4R Antibody alongside antibodies against potential partners

  • Apply secondary antibodies with complementary oligonucleotides

  • If proteins are in close proximity (<40 nm), ligation produces a circular DNA

  • Amplify signal through rolling circle amplification

  • Quantify fluorescent signals as measure of protein proximity

• Bioluminescence/Förster resonance energy transfer (BRET/FRET):

  • Create fusion constructs of MC4R and potential partners

  • Measure energy transfer as indicator of protein-protein interaction

  • Compare interaction efficiency between wild-type and mutant receptors

  • Assess how ligands affect heterodimer formation

• Functional consequences analysis:

  • Compare signaling properties of:

    • MC4R alone

    • Potential partner alone

    • Co-expressed receptors

  • Assess alterations in ligand potency, efficacy, or biased signaling

  • Determine if heterodimers have unique pharmacological profiles

This methodological framework enables researchers to explore how MC4R interactions with other receptors might influence its function in both physiological and pathological contexts.