TRPV3 Antibody, Biotin conjugated

What is TRPV3 and what are its primary biological functions?

TRPV3 (Transient Receptor Potential Cation Channel Subfamily V Member 3) is a non-selective cation channel that functions in various physiological processes, including temperature sensation and vasoregulation. It belongs to the TRP channel family that responds to diverse stimuli including temperature, pH, and various chemical ligands. TRPV3 is primarily expressed in skin keratinocytes and is activated by warm and hot temperatures between approximately 22-40°C. When activated, TRPV3 elevates intracellular calcium by facilitating a nonselective cationic conductance. The channel has been implicated in thermal sensation, skin barrier function, hair growth, and wound healing processes .

What is the molecular structure and localization of human TRPV3?

Human TRPV3 is encoded by a gene located on chromosome 17p13. The protein has an observed molecular weight of approximately 100 kDa, although its calculated molecular weight is 127,459 Da, suggesting potential post-translational modifications. The protein is classified under UniProt number Q8NET8 . TRPV3 is predominantly localized to the plasma membrane in normal physiological conditions, although it can also be found in intracellular compartments. Immunofluorescence studies have demonstrated significant co-localization of TRPV3 with Na⁺/K⁺ ATPase at the plasma membrane, confirming its surface expression. Under certain conditions, such as co-expression with TMEM79, TRPV3 may be redistributed to show broad cytoplasmic localization, including accumulation in the endoplasmic reticulum .

How should TRPV3 antibodies be stored and handled for optimal stability?

For optimal stability and performance, TRPV3 Antibody, Biotin conjugated should be stored at -20°C or -80°C upon receipt. Repeated freeze-thaw cycles should be strictly avoided as they can compromise antibody integrity and performance. The antibody is supplied in a storage buffer containing 0.03% Proclin 300 as a preservative, 50% Glycerol, and 0.01M PBS at pH 7.4 . When working with the antibody, it is recommended to aliquot the stock solution into smaller volumes before freezing to minimize freeze-thaw cycles. When handling the antibody for experiments, maintain cold chain practices by keeping it on ice or at 4°C. Prior to use, gently mix the antibody solution by inversion rather than vortexing to avoid protein denaturation .

What experimental applications is TRPV3 Antibody, Biotin conjugated suitable for?

TRPV3 Antibody, Biotin conjugated has been validated for several experimental applications including ELISA (Enzyme-Linked Immunosorbent Assay), which is its primary tested application . The biotin conjugation makes this antibody particularly suitable for detection systems employing streptavidin, which has an extremely high affinity for biotin. While specific validation data for this biotin-conjugated version may be limited to ELISA, other forms of TRPV3 antibodies have demonstrated utility in multiple applications including Western Blotting (WB), Immunohistochemistry (IHC), Immunocytochemistry (ICC), Immunofluorescence (IF), and Flow Cytometry . When adapting protocols from unconjugated antibodies, researchers should optimize detection conditions accounting for the biotin conjugation, potentially using streptavidin-horseradish peroxidase (HRP) or streptavidin-fluorophore conjugates for visualization .

What is the recommended protocol for using TRPV3 Antibody in immunohistochemistry?

For immunohistochemistry applications using TRPV3 antibody, the following optimized protocol has demonstrated successful results:

• Tissue preparation: Use paraffin-embedded tissue sections (TRPV3 antibodies have been validated on human mammary cancer and intestinal cancer tissues)

• Antigen retrieval: Perform heat-mediated antigen retrieval in citrate buffer (pH 6.0) for 20 minutes

• Blocking: Block tissue sections with 10% goat serum to reduce non-specific binding

• Primary antibody incubation: Incubate sections with TRPV3 antibody at a concentration of 1μg/ml overnight at 4°C

• Secondary antibody: Use biotinylated goat anti-rabbit IgG as secondary antibody with 30 minutes incubation at 37°C

• Detection system: Develop using Streptavidin-Biotin-Complex (SABC) with DAB as the chromogen

• Counterstaining: Counterstain with hematoxylin and mount with coverslip

While this protocol has been optimized for unconjugated TRPV3 antibody, the biotin-conjugated version would eliminate the need for biotinylated secondary antibody, allowing direct detection with streptavidin-HRP conjugate after primary antibody incubation .

How can TRPV3 Antibody, Biotin conjugated be used in flow cytometry applications?

For flow cytometry applications using TRPV3 antibody, follow this methodological approach:

• Cell preparation: Harvest cells of interest (validated cell lines include A431 and CACO-2 cells)

• Fixation: Fix cells with 4% paraformaldehyde to maintain cellular architecture

• Permeabilization: Treat cells with appropriate permeabilization buffer to facilitate intracellular staining

• Blocking: Block with 10% normal goat serum to reduce non-specific binding

• Primary antibody incubation: Incubate cells with TRPV3 antibody at a concentration of 1μg per 1×10⁶ cells for 30 minutes at 20°C

• Washing: Wash cells multiple times with buffer to remove unbound antibody

• Detection: For biotin-conjugated antibody, incubate with fluorophore-conjugated streptavidin

• Controls: Include appropriate isotype controls (rabbit IgG) and unstained samples

The biotin conjugation provides flexibility in detection strategy, allowing researchers to select from various fluorophore-conjugated streptavidin options (e.g., streptavidin-FITC, streptavidin-PE) depending on their flow cytometry setup and multicolor panel design .

How can TRPV3 Antibody be used to investigate TRPV3-TMEM79 interactions?

Investigation of TRPV3-TMEM79 interactions requires a multi-faceted approach combining several advanced techniques:

• Co-immunoprecipitation (Co-IP):

  • Transfect cells with tagged constructs (e.g., myc-TRPV3 and TMEM79-flag)

  • Lyse cells under non-denaturing conditions

  • Perform immunoprecipitation using anti-myc or anti-flag antibodies

  • Analyze precipitates by Western blotting with TRPV3 and TMEM79 antibodies

  • This approach has successfully demonstrated physical interaction between TRPV3 and TMEM79

• Proximity Ligation Assay (PLA):

  • Fix and permeabilize cells expressing both proteins

  • Incubate with primary antibodies against TRPV3 and TMEM79

  • Apply PLA probes and perform ligation and amplification

  • Visualize interaction as discrete fluorescent spots, indicating proteins within 40nm proximity

  • Include plasma membrane markers (e.g., Na⁺/K⁺ ATPase) to determine subcellular localization of interactions

• Immunofluorescence co-localization:

  • Use TRPV3 Antibody alongside TMEM79 antibodies in double immunofluorescence

  • Include organelle markers (e.g., calnexin for ER, LAMP-1 for lysosomes)

  • Analyze using confocal microscopy and quantify co-localization using appropriate software

  • This approach has revealed that TMEM79 causes redistribution of TRPV3 from plasma membrane to ER and lysosomal compartments

The biotin-conjugated TRPV3 antibody would be particularly useful in multi-labeling experiments, allowing versatile detection strategies in combination with other primary antibodies .

What methodologies can be used to study TRPV3 trafficking and degradation pathways?

Recent research has revealed complex mechanisms governing TRPV3 trafficking and degradation, particularly in relation to TMEM79. To investigate these processes, the following methodological approaches are recommended:

• Cell surface biotinylation assay:

  • Transfect cells with TRPV3 constructs (with/without TMEM79)

  • Label surface proteins with non-permeable biotin reagent

  • Isolate biotinylated proteins using streptavidin pull-down

  • Analyze TRPV3 levels by Western blotting

  • This approach can quantitatively assess plasma membrane expression of TRPV3

• Lysosomal vs. proteasomal degradation analysis:

  • Treat TRPV3-expressing cells with specific inhibitors:

    • Lysosomal inhibitors: Chloroquine (CQ), Bafilomycin A1 (BafA1)

    • Proteasomal inhibitors: MG132, Lactacystin

  • Analyze TRPV3 protein levels by Western blotting

  • Compare effects of different inhibitors to determine predominant degradation pathway

  • Research indicates TRPV3 is primarily degraded through the lysosomal pathway when co-expressed with TMEM79

• Protein synthesis and turnover assessment:

  • Perform cycloheximide (CHX) chase assay (100 μg/ml CHX treatment)

  • Collect samples at various timepoints (0, 3, 6, 12, 24 hours)

  • Analyze TRPV3 protein levels by Western blotting

  • Calculate protein half-life in different conditions

  • This approach has demonstrated accelerated degradation of TRPV3 when co-expressed with TMEM79

The biotin-conjugated TRPV3 antibody would be valuable for these experiments, particularly when combined with streptavidin-based detection systems for enhanced sensitivity .

How can electrophysiological methods be combined with TRPV3 immunodetection to correlate protein expression with channel function?

Integrating electrophysiological recordings with immunodetection techniques provides powerful insights into structure-function relationships of TRPV3 channels. The following comprehensive approach is recommended:

• Whole-cell patch-clamp recordings:

  • Prepare cells expressing TRPV3 (with/without regulatory proteins like TMEM79)

  • Record currents using voltage-clamp protocols with steps from -100 mV to +100 mV

  • Measure TRPV3 activation using specific agonists:

    • 2-APB (300 μM - 1 mM)

    • GSK 101 (1 μM)

  • Analyze current density (pA/pF) at different voltages (+60 mV, -60 mV)

  • Construct dose-response curves to determine EC50 values and potential shifts in agonist sensitivity

• Post-recording immunocytochemistry:

  • Mark recorded cells (e.g., with Lucifer Yellow in pipette solution)

  • Fix and immunostain using TRPV3 Antibody, Biotin conjugated

  • Detect with fluorophore-conjugated streptavidin

  • Quantify TRPV3 expression level/pattern in the same cell that was recorded

  • Correlate current density with protein expression level

• Calcium imaging with immunodetection:

  • Load cells with calcium indicators (Fura-2 AM or Fluo-4)

  • Measure calcium responses to TRPV3 agonists

  • Fix cells post-recording and perform immunostaining

  • Correlate calcium signal amplitude with TRPV3 expression pattern/level

This integrated approach has revealed that TMEM79 reduces TRPV3-mediated currents primarily by reducing surface expression rather than by altering channel properties .

What are the common challenges when using TRPV3 antibodies and how can they be overcome?

Researchers commonly encounter several challenges when working with TRPV3 antibodies, including the biotin-conjugated version. Here are methodological solutions to these issues:

• Limited antibody specificity:

  • Challenge: Search results note that "reliable anti-TRPV3 antibodies are hardly available"

  • Solution: Validate antibody specificity using:

    • Positive and negative control tissues/cells

    • TRPV3 knockout samples as negative controls

    • Epitope blocking experiments with immunizing peptide

    • Tagged TRPV3 constructs (e.g., myc-TRPV3) for cross-validation

• Weak signal in Western blotting:

  • Challenge: TRPV3 detection may show weak signal due to low expression or inefficient extraction

  • Solution:

    • Optimize protein extraction using specialized buffers containing 1% Triton X-100 or RIPA buffer

    • Include protease inhibitors to prevent degradation

    • For membrane proteins like TRPV3, extend transfer time or use specialized transfer systems

    • For biotin-conjugated antibody, use streptavidin-HRP with enhanced chemiluminescence detection

• High background in immunostaining:

  • Challenge: Biotin-conjugated antibodies may give high background due to endogenous biotin

  • Solution:

    • Block endogenous biotin using avidin/biotin blocking kit before antibody incubation

    • Optimize blocking conditions using 10% serum from the same species as secondary antibody

    • Include 0.1-0.3% Triton X-100 in blocking buffer for better penetration

    • Extensive washing with PBS containing 0.1% Tween-20

• Fixation-related epitope masking:

  • Challenge: Certain fixatives may mask the epitope recognized by TRPV3 antibody

  • Solution:

    • Compare different fixation methods (4% PFA, methanol, acetone)

    • Optimize antigen retrieval conditions (citrate buffer pH 6.0, EDTA buffer pH 9.0)

    • Adjust retrieval time and temperature

    • For tissues, prefer heat-mediated antigen retrieval in citrate buffer for 20 minutes

How can researchers optimize experimental conditions for detecting TRPV3 in different cellular compartments?

Detecting TRPV3 in different cellular compartments requires specialized approaches to overcome the challenges of membrane protein localization:

• Plasma membrane detection:

  • Co-stain with established plasma membrane markers (Na⁺/K⁺ ATPase, WGA, or membrane-targeted fluorescent proteins)

  • Use non-permeabilizing conditions for selective surface labeling

  • For live cell surface labeling, utilize cell-impermeable biotinylation reagents followed by fixation and streptavidin detection

  • Employ TIRF microscopy for selective visualization of plasma membrane TRPV3

• ER localization:

  • Co-stain with ER markers such as calnexin or PDI

  • Use gentle permeabilization (0.1% saponin rather than stronger detergents)

  • Optimize fixation using 4% PFA with 0.1% glutaraldehyde for better membrane preservation

  • Research has shown significant TRPV3 accumulation in the ER when co-expressed with TMEM79

• Lysosomal localization:

  • Co-label with lysosomal markers such as LAMP-1

  • Use pulse-chase approaches with fluorescently-tagged TRPV3 to track degradation

  • Pretreat cells with lysosomal inhibitors (chloroquine, bafilomycin A1) to enhance detection of transient lysosomal localization

  • Studies have demonstrated TRPV3 accumulation in lysosomes when co-expressed with TMEM79

• Quantitative assessment of compartmental distribution:

  • Perform subcellular fractionation followed by Western blotting with appropriate compartment markers

  • Use confocal microscopy with z-stack acquisition for 3D visualization

  • Employ quantitative image analysis with colocalization coefficients (Pearson's, Mander's)

  • For dynamic trafficking studies, use photoconvertible fluorescent protein tags

These optimized approaches have revealed that TMEM79 significantly alters the subcellular distribution of TRPV3, reducing plasma membrane localization and increasing ER and lysosomal accumulation .

How might TRPV3 antibodies be utilized to investigate its role in temperature sensation and skin disorders?

TRPV3 plays critical roles in temperature sensation and skin homeostasis, with emerging evidence linking its dysfunction to various skin disorders. Future research utilizing TRPV3 antibodies may focus on:

• Temperature-dependent conformational changes:

  • Use conformation-specific TRPV3 antibodies to detect structural changes upon temperature activation

  • Combine with FRET-based approaches to measure real-time conformational dynamics

  • Correlate structural changes with functional recordings in temperature-controlled environments

  • Develop experimental protocols to fix cells at specific temperatures to "capture" conformational states

• TRPV3 in skin barrier function:

  • Analyze TRPV3 expression patterns in normal vs. pathological skin samples

  • Correlate TRPV3 localization with markers of skin barrier integrity

  • Investigate potential changes in TRPV3-TMEM79 interactions in skin disorders

  • Examine TRPV3 expression in different skin cell populations (keratinocytes, immune cells, sensory neurons)

  • Research indicates that dysregulated TRPV3 may contribute to skin barrier dysfunction

• Therapeutic targeting strategies:

  • Use TRPV3 antibodies to screen for compounds that modulate channel trafficking

  • Develop assays to identify molecules that interfere with TRPV3-TMEM79 interaction

  • Establish high-throughput imaging approaches to monitor TRPV3 localization in response to potential therapeutic compounds

  • Combine with functional assays (calcium imaging, electrophysiology) to correlate trafficking with channel activity

The biotin-conjugated TRPV3 antibody would be particularly valuable for multiplexed detection approaches, allowing simultaneous visualization of TRPV3 alongside other proteins of interest in complex skin tissue samples .

What experimental approaches can be used to investigate the role of post-translational modifications in TRPV3 function?

Post-translational modifications (PTMs) likely play critical roles in regulating TRPV3 function, as suggested by the discrepancy between observed (100 kDa) and calculated (127 kDa) molecular weights. Future research methodologies to investigate PTMs include:

• Identification of PTM sites:

  • Immunoprecipitate TRPV3 using specific antibodies (including biotin-conjugated)

  • Perform mass spectrometry analysis to identify:

    • Phosphorylation sites (common regulatory mechanism for ion channels)

    • Glycosylation patterns (affecting membrane trafficking)

    • Ubiquitination sites (regulating degradation)

    • SUMOylation, acetylation, and other modifications

  • Compare PTM profiles between resting and stimulated conditions

• Functional impact assessment:

  • Generate TRPV3 mutants with abolished PTM sites

  • Combine site-directed mutagenesis with electrophysiological recordings

  • Use phospho-specific or other PTM-specific antibodies to correlate modifications with channel activity

  • Employ phosphatase or deglycosylation treatments to confirm PTM-dependent effects

• Dynamic regulation of PTMs:

  • Investigate how TMEM79 affects TRPV3 post-translational modifications

  • Examine whether temperature activation alters PTM patterns

  • Study PTM changes during trafficking between cellular compartments

  • Analyze whether degradation pathways are regulated by specific PTMs

Understanding these modifications will provide crucial insights into the molecular mechanisms governing TRPV3 function in normal physiology and disease states .

What are the most critical considerations for achieving reliable results with TRPV3 Antibody, Biotin conjugated?

To achieve reliable and reproducible results with TRPV3 Antibody, Biotin conjugated, researchers should consider several critical factors:

• Experimental validation and controls:

  • Always include appropriate positive and negative controls

  • Validate antibody specificity using TRPV3 knockout or knockdown samples

  • Include isotype controls to assess non-specific binding

  • For biotin-conjugated antibodies, include controls for endogenous biotin

  • Consider cross-validation with multiple TRPV3 antibodies recognizing different epitopes

• Storage and handling:

  • Strictly adhere to recommended storage conditions (-20°C or -80°C)

  • Avoid repeated freeze-thaw cycles by preparing small aliquots

  • Follow specific buffer recommendations (storage in 50% glycerol, 0.01M PBS, pH 7.4)

  • Maintain cold chain during experimental procedures

  • Check antibody stability and activity periodically

• Application-specific optimization:

  • Titrate antibody concentration for each specific application

  • Optimize fixation and permeabilization conditions for cellular localization studies

  • For biotin-conjugated antibodies, select appropriate streptavidin conjugates based on detection method

  • Consider the impact of tags or fusion proteins on epitope accessibility

  • Adjust incubation times and temperatures for optimal signal-to-noise ratio

• Data interpretation:

  • Consider potential redistribution of TRPV3 under different experimental conditions

  • Recognize that TRPV3 localization is dynamic and regulated by interacting proteins like TMEM79

  • Correlate immunodetection results with functional assays when possible

  • Be aware of potential differences between overexpression systems and endogenous expression

  • Report detailed methodological information to enable reproducibility

By carefully addressing these considerations, researchers can maximize the reliability and significance of their findings when using TRPV3 Antibody, Biotin conjugated in their investigations .

How does current TRPV3 research contribute to broader understanding of TRP channel biology and potential therapeutic applications?

Research utilizing TRPV3 antibodies contributes significantly to our understanding of TRP channel biology and therapeutic potential:

• Fundamental mechanisms of TRP channel regulation:

  • TRPV3-TMEM79 interaction studies reveal novel mechanisms of channel trafficking regulation

  • Investigations of temperature-dependent activation provide insights into thermosensation mechanisms

  • Studies of TRPV3 degradation pathways illuminate how ion channel turnover is regulated

  • These mechanisms may apply broadly to other TRP family members, advancing our understanding of sensory biology

• Physiological and pathological roles:

  • TRPV3 research contributes to understanding temperature sensation mechanisms

  • Studies of keratinocyte TRPV3 reveal roles in skin barrier function and homeostasis

  • Investigation of TRPV3 dysfunction may illuminate pathophysiology of skin disorders

  • Cross-talk between TRPV3 and other signaling pathways provides insights into integrated cellular responses

• Therapeutic targeting approaches:

  • Understanding TRPV3 regulation provides multiple intervention points:

    • Channel antagonists/agonists affecting gating properties

    • Compounds modulating protein-protein interactions (e.g., TRPV3-TMEM79)

    • Agents affecting trafficking pathways to alter surface expression

    • Interventions targeting specific post-translational modifications

  • These approaches could lead to treatments for conditions involving altered temperature sensation, pruritus, or skin barrier dysfunction

• Methodological advances:

  • Techniques developed for studying TRPV3, including the use of biotin-conjugated antibodies, contribute to broader methodological approaches for membrane protein research

  • Integrated approaches combining immunodetection with functional assays provide templates for investigating other ion channels

  • Advanced imaging and biochemical techniques for tracking protein trafficking and degradation have applications across cell biology