HMOX2 Antibody, FITC conjugated

What is HMOX2 and why is it important in research applications?

HMOX2 (Heme Oxygenase 2) is a 36 kDa constitutively expressed enzyme that catalyzes the degradation of heme to biliverdin, releasing carbon monoxide (CO) and free iron. Unlike its paralog HMOX1 (which is highly inducible under stress conditions), HMOX2 is constitutively expressed in mammalian tissues and plays a crucial role in producing carbon monoxide in the brain, where CO functions as a neurotransmitter . The CO signaling pathway parallels that of nitric oxide, highlighting HMOX2's importance in maintaining cellular homeostasis and neuroprotection. HMOX2 is involved in heme metabolism, leading to biliverdin and free iron generation, which are vital for cellular antioxidant defense mechanisms. Research applications focus on its role in neurobiology, heme homeostasis, and potential therapeutic applications in neurodegenerative diseases.

What applications are FITC-conjugated HMOX2 antibodies suitable for?

FITC-conjugated HMOX2 antibodies are primarily optimized for:

The FITC fluorophore has excitation/emission wavelengths of 499/515 nm, making it compatible with the 488 nm laser line commonly available in fluorescence microscopes and flow cytometers . While unconjugated HMOX2 antibodies can be used for Western Blot, IP, and IHC applications, the FITC-conjugated versions are specifically advantageous for direct visualization without requiring secondary antibodies.

How should I optimize immunofluorescence protocols for FITC-conjugated HMOX2 antibodies?

Optimizing immunofluorescence protocols for FITC-conjugated HMOX2 antibodies requires careful consideration of several parameters:

• Fixation method: For optimal HMOX2 detection, use 4% paraformaldehyde fixation for 15 minutes at room temperature. Over-fixation can mask epitopes.

• Permeabilization: Use 0.1-0.3% Triton X-100 for 5-10 minutes. HMOX2 is primarily cytoplasmic but may also localize to the endoplasmic reticulum.

• Blocking: Use 10% normal goat serum in PBS for 30-60 minutes at room temperature to minimize background fluorescence .

• Antibody concentration: Start with a 1:200-1:800 dilution and optimize from there. Based on flow cytometry studies, approximately 1 μg of antibody per 10^6 cells is often effective .

• Counterstains: DAPI works well for nuclear counterstaining without significant spectral overlap with FITC. For simultaneous visualization of multiple proteins, pair with far-red fluorophores to avoid bleed-through.

• Photobleaching prevention: Use anti-fade mounting media and minimize exposure to light during preparation and imaging.

An optimized protocol based on validated experiments includes tissue section blocking with 10% goat serum followed by incubation with rabbit anti-HMOX2 antibody (2-5 μg/mL) overnight at 4°C, which has been shown to provide specific staining in human lung squamous cell carcinoma and ovarian tissues .

What are the best methods for validating HMOX2 antibody specificity in my research model?

Validating HMOX2 antibody specificity is crucial for reliable experimental outcomes. Implement these methods:

• Positive and negative controls: Use known HMOX2-expressing cell lines (HepG2, K-562, HeLa, Jurkat) as positive controls . For negative controls, consider HMOX2 knockdown cells or tissues. Common positive controls from published literature include:

  Cell Line	HMOX2 Expression	Observed MW	Citation
  HepG2	High	36 kDa
  HeLa	Moderate	36-38 kDa
  Jurkat	Moderate	38 kDa
  DA3 mouse myeloma	High	43 kDa

• Peptide competition: Pre-incubate the antibody with excess recombinant HMOX2 protein before immunostaining to confirm signal specificity.

• Genetic validation: Compare staining in wild-type versus HMOX2 knockout/knockdown cells.

• Orthogonal methods: Validate protein expression with alternative methods (RT-PCR, Western blot) to confirm that fluorescence signal corresponds with other measures of HMOX2 expression.

• Cross-reactivity assessment: Test for potential cross-reactivity with HMOX1, which shares 42% amino acid sequence identity with HMOX2 . Ensure the antibody recognizes the intended target by comparing with known expression patterns.

In flow cytometry applications, MCF-7 cells have been validated for HMOX2 expression, showing specific staining when compared to isotype controls .

Why might I observe weak or absent signals when using FITC-conjugated HMOX2 antibodies?

Several factors can contribute to weak or absent signals with FITC-conjugated HMOX2 antibodies:

• Low protein expression: HMOX2 is constitutively expressed but may have variable expression levels across tissues. In cells where HMOX2 is minimally expressed, signal may be difficult to detect. Consider using signal amplification methods or more sensitive detection systems.

• Epitope masking: Improper fixation or processing can mask the epitope. Try different antigen retrieval methods—for IHC applications, EDTA buffer (pH 8.0) has shown better results than citrate buffer for HMOX2 detection .

• Photobleaching: FITC is susceptible to photobleaching. Minimize exposure to light during staining and use fresh anti-fade mounting media. Consider shorter exposure times with higher antibody concentrations.

• Buffer incompatibility: The storage buffer (typically PBS with 0.02% sodium azide and 50% glycerol, pH 7.3) may affect antibody performance. Ensure compatibility with your working buffers.

• Fluorophore:protein ratio: Suboptimal labeling can occur if the FITC:antibody ratio is too low. Commercial antibodies typically have a DOL (degree of labeling) of 3-5 FITC molecules per antibody.

For flow cytometry applications, where weak signals are common, researchers successfully used 1 μg of anti-HMOX2 antibody per 10^6 cells with a permeabilization step to access intracellular HMOX2, resulting in clear separation from control samples .

How can I minimize background and optimize signal-to-noise ratio in HMOX2 immunofluorescence?

Optimizing signal-to-noise ratio for FITC-conjugated HMOX2 antibodies requires systematic approaches:

• Enhanced blocking: Increase blocking time to 1-2 hours using a combination of 10% normal serum from the species of secondary antibody origin, 1% BSA, and 0.1% Tween-20 in PBS.

• Autofluorescence quenching: Tissue autofluorescence often overlaps with FITC emission. Treat sections with 0.1-1% sodium borohydride for 5 minutes, or use commercial autofluorescence quenchers before antibody application.

• Titration optimization: Determine the minimal antibody concentration that yields a positive signal. Based on validated protocols, 1:200-1:800 dilutions for IF/ICC applications provide optimal results .

• Buffer optimization: Use TBS instead of PBS if phosphate might interfere with binding. Add 0.05% Tween-20 to reduce non-specific hydrophobic interactions.

• Alternative counterstains: If DAPI/FITC crosstalk is an issue, consider using propidium iodide or far-red nuclear stains.

• Controls implementation: Always include an isotype control (rabbit IgG-FITC) to determine background levels. In flow cytometry, unstained samples and isotype controls help establish proper gating strategies .

• Spectral optimization: FITC has excitation/emission peaks at 499/515 nm. Ensure your filter sets are optimized for these wavelengths, with minimal bandwidth overlap with other channels .

For validated protocols, researchers successfully used anti-HMOX2 antibody at 2 μg/mL overnight at 4°C followed by thorough washing to achieve clear cellular localization patterns in MCF-7 cells while maintaining low background .

How can I use FITC-conjugated HMOX2 antibodies to investigate the relationship between heme binding and HMOX2 function?

Investigating heme binding and HMOX2 function using FITC-conjugated antibodies requires sophisticated experimental approaches:

• Co-localization studies: Use FITC-conjugated HMOX2 antibodies with red-shifted fluorophore-labeled heme sensors or heme transporters (like SLCO2B1) to visualize spatial relationships between heme and HMOX2. This approach has revealed that HMOX2 not only degrades heme but also functions as a heme buffer in cells.

• FRET analysis: For advanced studies, engineer proximity-based FRET pairs between FITC-HMOX2 antibodies and heme-binding proteins labeled with compatible acceptor fluorophores to monitor dynamic interactions in live cells.

• Pulse-chase experiments: Combine FITC-HMOX2 antibody labeling with pulsed introduction of fluorescently labeled heme analogs to track temporal dynamics of binding and processing.

• Oxidation state analysis: Recent research has shown that labile heme in cells exists predominantly in the ferric (Fe³⁺) state, with HMOX2 having differential binding affinities for ferric (Kᴅ = 3.6 nM) versus ferrous (Kᴅ = 320 nM) heme . This significant difference suggests HMOX2 may function as a buffer for oxidized heme.

• Mutational analysis: Label cells expressing wild-type versus heme-binding mutants of HMOX2 to correlate structural features with function.

Recent research using heme sensors with varying affinities demonstrated that HMOX2 significantly impacts cellular labile heme pools. When HEK293 cells were analyzed using flow cytometry with fluorescent heme sensors, they revealed approximately 50% heme occupancy of high-affinity sensors (HS1), which changed upon HMOX2 knockout or overexpression .

What are the most effective strategies for multiplexing FITC-conjugated HMOX2 antibodies with other markers in confocal microscopy?

Effective multiplexing with FITC-conjugated HMOX2 antibodies requires careful planning:

• Spectral compatibility: FITC (Ex/Em: 499/515 nm) pairs well with these fluorophores:

  Fluorophore	Excitation (nm)	Emission (nm)	Compatible Target
  DAPI/Hoechst	358	461	Nuclei
  Cy3	550	570	Membrane proteins
  Alexa Fluor 594	590	617	Organelle markers
  Alexa Fluor 647	650	665	Signaling proteins

• Sequential scanning: Use sequential rather than simultaneous scanning to avoid spectral bleed-through, particularly between FITC and other green-yellow fluorophores.

• Advanced microscopy techniques: Linear unmixing algorithms can separate overlapping emission spectra. For highest resolution, consider stimulated emission depletion (STED) microscopy, which works well with FITC conjugates.

• Combined protocols: For co-localization with HMOX1 (the inducible isoform), use FITC-HMOX2 with Cy5-conjugated HMOX1 antibodies to examine differential expression patterns in response to cellular stress.

• Live-dead discrimination: When using flow cytometry, combine FITC-HMOX2 with far-red dead cell markers to ensure analysis of viable populations only.

• Organelle targeting: Pair FITC-HMOX2 with mitochondrial (MitoTracker Red), ER (ER-Tracker Red), or Golgi (BODIPY TR ceramide) markers to determine subcellular localization patterns across different cell states.

Optimal antibody concentrations for multiplexing experiments are typically 2-5 μg/mL for FITC-HMOX2 combined with 1:500-1:1000 dilutions of commercial organelle dyes .

How do FITC-conjugated HMOX2 antibodies compare to other fluorophore conjugates for specific applications?

Different fluorophore conjugates offer distinct advantages depending on the application:

When tissue autofluorescence is a concern, far-red conjugates (APC or Alexa Fluor 647) provide better signal-to-noise ratios than FITC conjugates, particularly in tissues with high flavin or NADH content.

What are the critical differences in experimental design when using HMOX2 antibodies across various detection platforms?

Experimental design must be tailored to the detection platform when using HMOX2 antibodies:

• Western Blot vs. Immunofluorescence:

  • For Western blot: Denaturing conditions reveal linear epitopes, with HMOX2 typically detected at 36-43 kDa depending on the cell line

  • For IF: Native conformation preservation is critical, requiring gentler fixation (4% PFA rather than methanol)

• Flow Cytometry vs. Microscopy:

  • Flow cytometry: Higher antibody concentrations (1 μg/10^6 cells) are needed for sufficient signal

  • Microscopy: Lower concentrations (1:200-1:800 dilutions) with longer incubation times provide better signal-to-noise ratios

• IHC Protocol Adaptations:

  • When using HMOX2 antibodies for IHC, EDTA buffer (pH 8.0) typically provides better epitope retrieval than citrate buffer (pH 6.0)

  • Paraffin-embedded sections require more rigorous antigen retrieval than frozen sections

• Live Cell vs. Fixed Cell Analysis:

  • Fixed cell analysis: Standard FITC-conjugated HMOX2 antibodies work well following permeabilization

  • Live cell analysis: Requires cell-permeable antibody fragments or alternative approaches like fluorescent protein fusions

• Cross-Platform Validation:

  • When comparing HMOX2 expression across platforms, recognize that flow cytometry provides population averages while IF shows individual cell heterogeneity

  • Western blot can validate antibody specificity through molecular weight confirmation, showing HMOX2 at approximately 36 kDa

For detection of mouse HMOX2, researchers used Simple Western™ and found that the relative molecular weight appears slightly higher (43 kDa) than in human samples (36-38 kDa), highlighting the importance of species-specific optimization .

How can FITC-conjugated HMOX2 antibodies be used to investigate labile heme dynamics in cellular stress responses?

FITC-conjugated HMOX2 antibodies offer unique opportunities to study labile heme dynamics during stress:

• Dual labeling approaches: Combine FITC-HMOX2 antibodies with genetically encoded fluorescent heme sensors (like HS1) to simultaneously track HMOX2 localization and labile heme pools. Recent research demonstrated that HEK293 cells maintain labile heme at approximately 50% occupancy of high-affinity sensors, which is affected by HMOX2 expression levels .

• Stress response kinetics: Track the temporal relationship between oxidative stress induction, HMOX2 redistribution, and changes in labile heme using time-lapse imaging with FITC-HMOX2 antibodies in permeabilized cells.

• Cellular compartmentalization: Recent research suggests HMOX2 predominantly binds ferric (Fe³⁺) rather than ferrous (Fe²⁺) heme, with dramatically different dissociation constants (3.6 nM vs. 320 nM respectively) . This can be investigated using FITC-HMOX2 antibodies combined with oxidation state-specific heme probes.

• Competitive binding studies: Use flow cytometry with FITC-HMOX2 antibodies in cells expressing varying levels of other heme-binding proteins to determine hierarchy of heme distribution during stress conditions.

• Microenvironmental changes: Explore how pH fluctuations during cellular stress affect HMOX2-heme interactions, considering that FITC fluorescence is pH-sensitive and can serve as an indirect pH indicator in well-controlled experiments.

Mathematical modeling based on recent work indicates that the competition between HMOX2 and heme sensors for labile heme follows specific equilibria, where factors including the competition constant (KComp) and concentrations of HMOX2 (approximately 10 nM in cells) significantly impact experimental outcomes .

What role could FITC-conjugated HMOX2 antibodies play in investigating neurodegenerative disease mechanisms?

FITC-conjugated HMOX2 antibodies offer valuable tools for neurodegenerative disease research:

• Neural CO signaling: HMOX2 produces carbon monoxide (CO) which functions as a neurotransmitter . Using FITC-HMOX2 antibodies in conjunction with CO-sensitive probes could illuminate disruptions in this signaling pathway in neurodegenerative conditions.

• Blood-brain barrier studies: Apply FITC-HMOX2 antibodies in brain endothelial cell models to investigate how HMOX2 expression and localization correlate with barrier integrity in neurodegenerative conditions.

• Oxidative stress visualization: HMOX2's role in handling oxidized (ferric) heme makes it relevant to neurodegenerative conditions associated with oxidative stress. FITC-HMOX2 antibodies can track redistribution of this enzyme during disease progression.

• Cellular protection mechanisms: HMOX2 activity leads to production of biliverdin (subsequently converted to bilirubin), which has antioxidant properties. Visualizing HMOX2 in relation to markers of cellular damage could reveal protective mechanisms.

• Differential expression analysis: Compare HMOX2 versus HMOX1 expression patterns in neurodegeneration using multiplexed immunofluorescence. While HMOX1 is inducible under stress, HMOX2 is constitutively expressed and may play different roles in disease progression.

• Therapeutic target assessment: FITC-HMOX2 antibodies can help evaluate the efficacy of therapeutic interventions targeting heme metabolism in neurodegenerative conditions by monitoring changes in HMOX2 localization or expression.