TRPM3 Antibody, FITC conjugated

What is TRPM3 and why is it important in research?

TRPM3 (Transient Receptor Potential Melastatin 3) is a calcium-permeable ion channel belonging to the TRP family of cation channels. These channels are critical for cellular calcium signaling and homeostasis. The protein encoded by the TRPM3 gene mediates calcium entry, which is potentiated by calcium store depletion . TRPM3 has emerged as an important research target for several reasons:

• It is ubiquitously expressed in multiple cell types, including vascular smooth muscle cells (VSMCs) and immune cells like natural killer (NK) cells and B lymphocytes

• It can be activated by the neurosteroid pregnenolone sulphate and sphingosine, making it a target for studying steroid-mediated signaling

• It has been implicated in various physiological processes and pathological conditions, including vascular biology and chronic fatigue syndrome/myalgic encephalomyelitis (CFS/ME)

The functional importance of TRPM3 makes it a valuable target for researchers investigating calcium signaling pathways, ion channel pharmacology, and disease mechanisms.

What applications are FITC-conjugated TRPM3 antibodies suitable for?

FITC-conjugated TRPM3 antibodies are particularly valuable for fluorescence-based applications. Based on available product information and research studies, these antibodies are suitable for:

• Immunofluorescence (IF): FITC-conjugated antibodies enable direct visualization of TRPM3 protein in fixed cells without requiring a secondary antibody

• Flow cytometry: For quantitative measurement of TRPM3 surface expression on cells like NK cells and B lymphocytes

• Immunohistochemistry with fluorescence detection (IF/IHC-P): For localizing TRPM3 in tissue sections while preserving spatial information

• Live cell imaging: Some antibodies targeting extracellular epitopes may be suitable for non-permeabilized, live cell applications

When designing experiments, researchers should verify the specific applications validated for their particular FITC-conjugated TRPM3 antibody, as performance can vary between products from different manufacturers.

How should FITC-conjugated TRPM3 antibodies be stored for optimal performance?

Proper storage is critical for maintaining the functionality of FITC-conjugated antibodies. According to product information:

• Short-term storage (up to 12 months): Store at 4°C in light-protected vials or covered with light-protecting material (e.g., aluminum foil)

• Long-term storage (up to 24 months): Dilute with up to 50% glycerol and store at -20°C to -80°C

• Shipping condition: Typically shipped at 4°C

• Freeze-thaw cycles: Avoid repeated freeze/thaw cycles as they will compromise both enzyme activity and antibody binding

• Buffer composition: Typically stored in buffers like 0.01M TBS (pH 7.4) with 1% BSA, 0.02% Proclin300, and 50% Glycerol

Light protection is particularly important for FITC-conjugated antibodies, as fluorescein is susceptible to photobleaching when exposed to light for extended periods.

How can I validate the specificity of a TRPM3 antibody in my experimental system?

Validating antibody specificity is crucial for ensuring reliable experimental results. Several approaches have been documented in TRPM3 research:

• siRNA knockdown: Transfect cells with TRPM3-specific siRNA and confirm reduced antibody labeling compared to control siRNA. This approach has been successfully used to validate TRPM3 antibodies in vascular smooth muscle cells .

• Pre-absorption controls: Pre-incubate the antibody with its antigenic peptide (e.g., 10 μM) before applying to cells or tissues. Specific binding should be eliminated or significantly reduced, as demonstrated in studies with TM3E3 antibody .

• Inducible expression systems: Use tetracycline-inducible TRPM3 expression systems (Tet+ vs. Tet-) to demonstrate increased antibody binding following TRPM3 induction .

• Negative controls: Include primary antibody omission controls in immunofluorescence experiments to assess background and non-specific secondary antibody binding .

• Positive controls: Test the antibody on tissues or cell types known to express TRPM3, such as NK cells or B lymphocytes .

The combination of these approaches provides strong evidence for antibody specificity and helps rule out potential false-positive results.

What are the optimal fixation and permeabilization protocols for TRPM3 immunodetection?

Optimal fixation and permeabilization are critical for preserving TRPM3 epitopes while allowing antibody access. Based on published protocols:

For immunofluorescence labeling:

• Fix cells in 2% paraformaldehyde for 5 minutes at room temperature

• For antibodies targeting intracellular epitopes, permeabilization is required (though specific permeabilization agents were not detailed in the search results)

• Incubate with primary antibody (e.g., 1:1000 dilution) overnight at 4°C

• Incubate with fluorescent secondary antibody (if using non-conjugated primary) for 2 hours at room temperature

For antibodies targeting extracellular epitopes like TM3E3:

• These can be applied to live, non-permeabilized cells in culture medium during pre-incubation protocols

• This approach allows detection of surface-expressed TRPM3 channels and can be confirmed by immunocytochemistry

The specific protocol should be optimized based on your particular antibody, sample type, and application requirements.

How can TRPM3 antibodies be used to study channel functionality in live cells?

TRPM3 antibodies, particularly those targeting extracellular epitopes, have been successfully used to study channel functionality in live cells:

• Blocking antibody approach: The TM3E3 antibody, developed against a conserved peptide of the third extracellular loop of TRPM3, has been shown to partially inhibit TRPM3 channel function when applied extracellularly. This approach has enabled:

  • Inhibition of pregnenolone sulphate-evoked currents in patch-clamp recordings

  • Reduction of calcium entry in TRPM3-expressing cells

  • Blockade of sphingosine-activated TRPM3 channels

• Pre-incubation protocols: Antibodies can be added to cell culture medium during pre-incubation periods, allowing them to bind to TRPM3 and remain bound during subsequent functional assays even after the antibody is removed from the recording medium .

• Combined with calcium imaging: TRPM3 channel function can be assessed by measuring intracellular calcium using fluorescent indicators like Fura-2 AM (cytoplasmic calcium) and Rhod-2 AM (mitochondrial calcium) in combination with antibody treatments .

This approach has revealed that TRPM3 antibodies can provide isoform-specific blockade of channel function, offering advantages over small-molecule inhibitors that may lack specificity.

What controls should be used when working with FITC-conjugated TRPM3 antibodies?

When working with FITC-conjugated TRPM3 antibodies, proper controls are essential for experimental rigor:

• Isotype controls: Include a FITC-conjugated isotype control antibody (same host species and isotype, e.g., rabbit IgG-FITC) to assess background fluorescence and non-specific binding.

• Secondary antibody-only controls: When used alongside unconjugated primary antibodies in the same experiment, include samples treated with secondary antibody only.

• Antigen pre-absorption controls: Pre-incubate the antibody with its antigenic peptide before staining to demonstrate binding specificity .

• Negative cell/tissue controls: Include samples known to express low or undetectable levels of TRPM3 to establish background signal levels.

• Positive cell/tissue controls: Include samples with confirmed TRPM3 expression, such as tetracycline-induced TRPM3-expressing cells .

• Auto-fluorescence controls: Include unstained samples to assess natural tissue auto-fluorescence, particularly important when working with tissues rich in auto-fluorescent components.

• Cross-reactivity controls: If conducting multiplex experiments, validate that other antibodies in your panel do not cross-react with the TRPM3 antibody.

These controls help establish the specificity of the observed signals and enable accurate interpretation of experimental results.

What are the considerations for using TRPM3 antibodies in tissue-specific studies?

When using TRPM3 antibodies for tissue-specific studies, several important considerations must be addressed:

• Expression pattern variations: TRPM3 expression varies across tissues. For example, studies have shown differential expression between vascular smooth muscle cells (VSMCs) and endothelial cells, with VSMCs showing higher expression levels . Experimental design should account for these tissue-specific differences.

• Epitope conservation: Consider the conservation of the target epitope across species when studying animal tissues. For example, the TM3E3 antibody targets a peptide (CLFPNEEPSWKLAKN) that is completely conserved in human, mouse, and rat, making it suitable for cross-species studies .

• Tissue preparation: Different tissues may require optimized fixation protocols to preserve TRPM3 epitopes while maintaining tissue morphology.

• Cell type identification: Combine TRPM3 antibody staining with cell-type-specific markers to identify which cells express TRPM3 within heterogeneous tissues. This approach has been used to identify TRPM3 in specific NK cell subsets (CD56Bright vs. CD56Dim) and B lymphocytes (CD19+) .

• Functional state: Consider that TRPM3 expression may vary depending on the functional state of cells. For example, TRPM3 has been detected in both contractile and proliferating VSMCs .

• Pathological conditions: TRPM3 expression may be altered in disease states, as observed in chronic fatigue syndrome/myalgic encephalomyelitis, where decreased TRPM3 expression was found in NK cells and B lymphocytes .

These considerations help ensure accurate characterization of TRPM3 expression and function in specific tissue contexts.

How do different TRPM3 antibodies compare in terms of epitope recognition and specificity?

TRPM3 antibodies target different epitopes of the channel protein, which affects their applications and specificity:

Key observations about epitope targeting:

• Extracellular domain antibodies (like TM3E3) can be used for functional studies on live cells and have been successful in blocking channel activity .

• Immunogen design affects specificity - antibodies generated against highly conserved regions show cross-species reactivity, while those against divergent regions may be species-specific .

• Functional impact varies - TM3E3 produces approximately 50% blockade of channel function, similar to other E3-targeted antibodies against six-transmembrane type ion channels .

• Cross-reactivity testing is important - TM3E3 showed no effect on related channels TRPC5, TRPV4, TRPM2, or endogenous ATP responses, demonstrating specificity .

When selecting a TRPM3 antibody, researchers should consider these differences in epitope recognition and choose antibodies validated for their specific application and species of interest.

How can I troubleshoot weak or inconsistent TRPM3 antibody signals in my experiments?

When facing challenges with TRPM3 antibody signals, systematic troubleshooting approaches can help identify and resolve issues:

• Sample preparation issues:

  • Optimize fixation time and conditions - overfixation can mask epitopes

  • Ensure adequate permeabilization for antibodies targeting intracellular domains

  • Try antigen retrieval methods if working with paraffin-embedded tissues

• Antibody-related factors:

  • Titrate antibody concentration - try dilutions ranging from 1:50 to 1:5000 based on application

  • Consider antibody storage conditions - FITC conjugates are light-sensitive and may deteriorate if improperly stored

  • Test alternative TRPM3 antibodies targeting different epitopes

• Expression level considerations:

  • TRPM3 expression varies between tissues and cell types - endothelial cells show less TRPM3 mRNA compared to VSMCs

  • Expression may be altered in disease states - decreased in certain immune cells in CFS/ME patients

  • Consider using positive controls with confirmed high TRPM3 expression

• Technical optimizations:

  • Extend primary antibody incubation time (overnight at 4°C instead of shorter incubations)

  • Modify blocking solutions to reduce background while preserving specific signals

  • For FITC-conjugated antibodies, protect from light during all steps and minimize exposure time during imaging

• Functional verification:

  • Combine antibody detection with functional assays (e.g., calcium influx measurements) to confirm channel expression

  • Use siRNA knockdown to validate signals (although complete knockdown may be challenging, as noted in studies where "only partial knock-down of the pregnenolone sulphate response could be achieved")

By systematically addressing these potential issues, researchers can improve the reliability and consistency of TRPM3 antibody signals in their experiments.

What approaches can be used to study TRPM3 channel dynamics using antibody-based methods?

Several sophisticated approaches utilizing antibodies can reveal TRPM3 channel dynamics:

• Antibody-based channel inhibition with calcium imaging:

  • Pre-incubate cells with TRPM3 antibodies targeting extracellular domains (like TM3E3)

  • Stimulate with TRPM3 agonists (pregnenolone sulphate or sphingosine)

  • Measure changes in intracellular calcium using fluorescent indicators like Fura-2 AM

  • This approach revealed that TM3E3 partially inhibits calcium entry independently of the agonist used

• Combining electrophysiology with antibody treatments:

  • Apply antibodies during whole-cell patch-clamp recordings

  • Monitor changes in current amplitude and I-V relationships over time

  • This approach showed that TM3E3 caused partial reduction in TRPM3 currents over 5-10 minutes

• Antibody-based detection of surface expression changes:

  • Use flow cytometry with extracellular-targeting antibodies to quantify surface TRPM3

  • Apply stimuli that may alter trafficking or expression

  • This approach revealed decreased TRPM3 expression on CD56Bright NK cells and CD19+ B cells in CFS/ME patients

• Correlation of TRPM3 expression with functional responses:

  • Compare TRPM3 antibody labeling intensity with magnitude of functional responses

  • Link expression levels to channel activity in different conditions

  • This approach showed correlation between reduced TRPM3 expression and decreased calcium influx in immune cells from CFS/ME patients

• Antibody-based isolation of TRPM3 complexes:

  • Use antibodies for immunoprecipitation to isolate TRPM3 and associated proteins

  • Identify interaction partners that may regulate channel function

  • While not explicitly described in the search results, this is a standard approach for studying ion channel protein complexes

These approaches provide complementary information about TRPM3 channel expression, localization, and functional properties in various experimental contexts.

What are the effects of TRPM3 antibodies on gene expression regulation?

Research has shown that TRPM3 antibodies can indirectly affect gene expression through modulation of calcium signaling pathways:

• HSP70 expression regulation:

  • Studies have demonstrated that TRPM3 activation by pregnenolone sulfate (PS) can upregulate HSP70 mRNA expression approximately 2-fold

  • When PS was co-administered with mefenamic acid (MFA, a TRPM3 inhibitor), this modulation of HSP70 mRNA was lost

  • While not directly tested with antibodies in the search results, TRPM3-blocking antibodies like TM3E3 would be expected to produce similar effects by inhibiting TRPM3-mediated calcium entry

• Calcium-dependent transcription:

  • TRPM3 mediates calcium entry, which can activate calcium-dependent transcription factors

  • By partially blocking TRPM3 channels, antibodies like TM3E3 may modulate these calcium-dependent signaling pathways

  • This suggests potential applications for TRPM3 antibodies in studying the role of calcium signaling in gene regulation

• Cell signaling in immune cells:

  • In studies of chronic fatigue syndrome/myalgic encephalomyelitis, reduced TRPM3 expression correlated with decreased calcium mobilization during receptor cross-linking

  • This suggests TRPM3 plays a role in calcium-dependent immune cell signaling and potentially gene expression regulation in pathological conditions