TRPM3 Antibody, HRP conjugated

What is the molecular target of TRPM3 antibodies?

TRPM3 belongs to the family of transient receptor potential (TRP) channels that function as cation-selective channels critical for cellular calcium signaling and homeostasis. The protein encoded by the TRPM3 gene (80036) mediates calcium entry, which can be potentiated by calcium store depletion. Alternatively spliced transcript variants encoding different isoforms have been identified . TRPM3 is also known by several synonyms including GON-2, LTRPC3, and Melastatin-2, with a molecular mass of approximately 197.6 kDa .

What are the principal applications of HRP-conjugated TRPM3 antibodies?

HRP-conjugated TRPM3 antibodies are primarily utilized in:

Unlike unconjugated antibodies that require a secondary detection system, HRP-conjugated antibodies enable direct visualization of target proteins, simplifying experimental workflows and potentially reducing background signal .

How do storage conditions affect HRP-conjugated TRPM3 antibody performance?

Proper storage is critical for maintaining the functional integrity of both the antibody binding domain and the enzymatic activity of the HRP conjugate:

• Storage temperature: Store at -20°C

• Preparation: Aliquot into multiple vials to avoid repeated freeze-thaw cycles

• Buffer composition: Typically maintained in aqueous buffered solution containing 0.01M TBS (pH 7.4) with 1% BSA, 0.03% Proclin300, and 50% Glycerol

Repeated freeze-thaw cycles significantly compromise both binding affinity and enzymatic activity, leading to diminished signal intensity and increased background .

How should experimental controls be designed when working with TRPM3 antibodies?

Proper experimental design requires multiple control strategies:

• Negative controls:

  • Omission of primary antibody to assess secondary antibody specificity

  • Use of isotype-matched irrelevant antibodies to evaluate non-specific binding

  • Samples known to lack TRPM3 expression (validated by alternative methods)

• Positive controls:

  • HEK293 cells overexpressing TRPM3 (widely used as positive controls)

  • Human saphenous vein smooth muscle cells (HSV SMC) which naturally express TRPM3

• Blocking peptide controls:

  • Pre-absorption of antibody with immunizing peptide to confirm specificity

  • Parallel staining with antibodies targeting different TRPM3 epitopes

Experimental validation data demonstrates that cells lacking TRPM3 cDNA transfection show no response to TRPM3 agonists like pregnenolone sulfate, confirming specificity .

What are the optimal sample preparation methods for TRPM3 detection using HRP-conjugated antibodies?

Sample preparation protocols should be optimized based on the specific application:

For Western Blotting:

• Homogenization in 50 mM Tris (pH 7.4) containing 0.2% CHAPS and protease inhibitor cocktail

• Incubation for 2 hours at 4°C followed by clarification by centrifugation at 18,000 ×g

• Proper denaturation at 95°C for 5 minutes in reducing sample buffer

For Immunohistochemistry:

• Formalin-fixed, paraffin-embedded tissues require antigen retrieval

• After routine deparaffinization, rehydration, and blocking, tissues should undergo appropriate antigen retrieval procedures

• Overnight incubation at 4°C with primary TRPM3 antibody followed by visualization procedures

How can non-specific background be minimized when using HRP-conjugated TRPM3 antibodies?

Non-specific background represents a common challenge with HRP-conjugated antibodies. Optimization strategies include:

• Blocking optimization:

  • Extend blocking time to 1-2 hours at room temperature

  • Test different blocking agents (BSA, normal serum, commercial blockers)

  • Include 0.1-0.3% Triton X-100 for membrane permeabilization when detecting intracellular epitopes

• Antibody dilution:

  • Perform careful titration experiments to determine optimal concentration

  • Consider using lower antibody concentrations (1:1000-5000) for Western blotting applications

  • Higher dilutions (1:300-500) may be necessary for IHC applications

• Washing procedures:

  • Increase wash durations and frequencies between steps

  • Include 0.05% Tween-20 in wash buffers to reduce non-specific hydrophobic interactions

What factors affect epitope recognition by different TRPM3 antibodies?

TRPM3 antibodies target different epitopes across the protein structure, which affects their experimental utility:

Different epitopes may be differentially accessible depending on protein conformation, fixation methods, and experimental conditions .

How can HRP-conjugated TRPM3 antibodies be used to investigate channel modulation by signaling pathways?

TRPM3 channels are regulated by complex signaling mechanisms that can be investigated using antibodies:

• G-protein coupled receptor (GPCR) modulation:

  • μ-opioid receptors inhibit TRPM3 via Gβγ subunits

  • G protein βγ subunits directly inhibit TRPM3, while Gα subunits do not affect channel activity

  • Multiple Gαi-coupled GPCRs (GABAB, CB2, δ-opioid, adrenoreceptors) inhibit TRPM3

• Protein kinase regulation:

  • Protein kinase CK2 co-immunoprecipitates with TRPM3 and phosphorylates the channel

  • CK2-mediated phosphorylation controls TRPM3 channel activity in INS-1 β-cells

  • Phosphorylation can be detected using phosphospecific antibodies that recognize CK2 substrate motifs

• Steroid hormone interactions:

  • Pregnenolone sulfate activates TRPM3 while progesterone inhibits channel activity

  • Binding can be assessed using steroid overlay assays with TRPM3 immunoprecipitates

Experimental approaches combining electrophysiology with immunological detection provide powerful tools for investigating these regulatory mechanisms.

What are the methodological approaches for investigating TRPM3 involvement in disease pathophysiology?

TRPM3 has been implicated in several pathophysiological processes, including kidney cancer and vascular disorders. Research methodologies include:

• Expression analysis in disease tissues:

  • IHC analysis of TRPM3 protein in tumor microarrays

  • qPCR for TRPM3 mRNA expression between paired tumor-normal tissues

  • Combined approaches revealed that TRPM3 expression is lower in kidney cancer (KIRC) tumor tissues compared to normal tissues

• Functional studies in disease models:

  • Calcium imaging to assess TRPM3-mediated calcium responses in cells derived from disease tissues

  • Inhibition studies using specific TRPM3 blockers like TM3E3 (E3-targeting antibody)

  • Assessment of disease-relevant endpoints (e.g., IL-6 and MMP-9 secretion in vascular cells)

• Sex differences in TRPM3 function:

  • Wire myography to analyze vasoactive responses in human coronary and meningeal arteries

  • Comparison of TRPM3 mRNA and protein expression between males and females

  • Studies have revealed enhanced pregnenolone sulfate-induced relaxation in arteries from females compared to males

How can researchers validate the specificity of TRPM3 antibody signals?

Multiple complementary approaches should be employed to ensure specificity:

• Genetic validation:

  • Comparison of antibody signals in wild-type versus TRPM3 knockout models

  • siRNA knockdown of TRPM3 followed by antibody detection

  • Heterologous expression systems (e.g., tetracycline-inducible TRPM3 cell lines)

• Pharmacological validation:

  • Correlation of antibody detection with functional responses to TRPM3 agonists (pregnenolone sulfate, nifedipine)

  • Blockade with specific TRPM3 antagonists (isosakuranetin)

  • Comparison with selective antibodies targeting different epitopes

• Technical validation:

  • Peptide competition assays to confirm epitope specificity

  • Comparison across multiple detection methods (WB, IHC, IF)

  • Mass spectrometry validation of immunoprecipitated proteins

What approaches can resolve contradictory findings when using different TRPM3 antibodies?

Discrepancies between studies using different TRPM3 antibodies can arise from multiple factors:

• Splice variant detection:

  • Human TRPM3 exists in multiple splice variants

  • Antibodies targeting different regions may detect distinct subsets of variants

  • Solution: Use RT-PCR with isoform-specific primers to characterize expressed variants in the experimental system

• Post-translational modifications:

  • Phosphorylation by CK2 affects TRPM3 channel activity

  • Some antibodies may have differential sensitivity to phosphorylated epitopes

  • Solution: Use phosphorylation-specific antibodies or phosphatase treatment prior to detection

• Cross-reactivity issues:

  • Some antibodies may detect related TRP family members

  • Solution: Validate using overexpression systems, knockout controls, and multiple antibodies targeting different epitopes

When confronted with contradictory findings, researchers should carefully document antibody characteristics, including catalog numbers, host species, epitope regions, and validation methods employed.