PRODH Antibody, FITC conjugated

What is PRODH and why is it important in research?

PRODH, or Proline Dehydrogenase 1, is a mitochondrial oxidoreductase involved in the transfer of redox potential across the mitochondrial membrane. Also known under alternate names including PIG6, HSPOX2, PRODH1, PRODH2, POX, SCZD4, and TP53I6, PRODH belongs to the proline oxidase family . Its significance in research stems from its role in cellular metabolism and potential implications in various pathological conditions. The protein has a calculated molecular weight of 68 kDa (600 amino acids), though it is typically observed at 56 kDa and 66 kDa in Western blot applications .

Understanding PRODH function requires reliable antibody detection systems, making properly characterized antibodies crucial for advancing research in this field. The protein's mitochondrial localization and enzymatic function make it particularly interesting for studies of cellular metabolism, stress responses, and related disorders.

What are the key characteristics of commercially available PRODH antibodies?

PRODH antibodies are available with various specifications tailored to different research applications. Typically, these antibodies target specific amino acid sequences within the PRODH protein. For instance, some antibodies target amino acids 71-134, while others target regions like 130-155, 141-240, or 441-540 .

The most common host species is rabbit, with polyclonal antibodies being widely available . The observed reactivity is primarily with human, mouse, and rat samples. Key specifications include:

Different PRODH antibodies show varied application suitability, with some optimized for Western blotting while others perform better in immunohistochemistry or immunofluorescence applications .

How should PRODH antibodies be stored and handled for optimal performance?

PRODH antibodies require specific storage conditions to maintain their functionality. The typical storage recommendation is -20°C, with antibodies remaining stable for approximately one year after shipment . Many commercial preparations are supplied in PBS buffer containing 0.02% sodium azide and 50% glycerol at pH 7.3, which helps prevent freezing damage and maintains antibody stability .

For small aliquots (e.g., 20μl), some manufacturers add 0.1% BSA to further stabilize the antibody . Importantly, repeated freeze-thaw cycles should be avoided to preserve antibody performance. While some suppliers note that aliquoting is unnecessary for -20°C storage, this practice may still be beneficial for antibodies that will be used multiple times over an extended period.

Upon receipt, antibodies should be immediately stored according to manufacturer recommendations, and working dilutions should be prepared fresh before each experiment to ensure optimal binding and specificity.

How does FITC conjugation affect antibody binding characteristics?

FITC (Fluorescein isothiocyanate) conjugation can significantly impact antibody performance. Research has demonstrated that the FITC-labeling index (number of FITC molecules per antibody) is negatively correlated with binding affinity for target antigens . This is a critical consideration for researchers using FITC-labeled antibodies as primary detection tools.

The binding affinity reduction occurs because FITC molecules may attach to amino acid residues within or near the antigen-binding regions of the antibody, potentially interfering with the antibody-antigen interaction. This modification of binding properties has important implications for experimental design and interpretation.

While higher FITC-labeling indices generally result in brighter fluorescence signals, this increased sensitivity comes at the cost of reduced specificity and potentially increased non-specific background staining . Therefore, researchers must carefully balance detection sensitivity with binding specificity when selecting FITC-conjugated antibodies for their experiments.

What is the optimal FITC-labeling index for research applications?

The optimal FITC-labeling index depends on the specific research application and must balance sensitivity with specificity. Based on published findings, researchers should consider testing several antibodies with different labeling indices to identify the most suitable for their specific application .

For tissue cross-reactivity (TCR) studies and applications requiring high specificity, an antibody with a moderate labeling index is generally preferred to minimize the decrease in binding affinity while maintaining adequate fluorescence intensity . This approach helps achieve appropriate sensitivity while reducing non-specific staining.

Researchers should request information about the FITC-labeling index from manufacturers or consider performing comparative analyses when multiple labeled antibodies are available. This careful selection process is essential for obtaining reliable and reproducible results, particularly in applications like immunohistochemistry where interpretation of staining patterns is critical.

What controls should be implemented when using FITC-conjugated PRODH antibodies?

Implementing proper controls is essential when using FITC-conjugated PRODH antibodies to ensure result validity. The following controls should be considered:

• Isotype controls: Include a FITC-conjugated antibody of the same isotype (e.g., Rabbit IgG-FITC) but without specificity for PRODH to assess non-specific binding.

• Blocking controls: Pre-incubate sections with unlabeled PRODH antibody before applying the FITC-conjugated antibody to demonstrate binding specificity.

• Absorption controls: Pre-incubate the FITC-PRODH antibody with recombinant PRODH protein before application to validate specificity.

• Autofluorescence controls: Examine unstained samples to identify any inherent tissue autofluorescence that could interfere with FITC signal interpretation.

• Secondary-only controls: For indirect detection methods, include samples treated only with secondary reagents to identify non-specific binding of detection components.

Additionally, researchers should include positive control tissues known to express PRODH (e.g., liver tissue, cerebellum) and negative control tissues to validate staining patterns . These comprehensive controls help distinguish true positive signals from technical artifacts and ensure reliable data interpretation.

What are the optimal protocols for different applications of PRODH-FITC antibodies?

The optimal protocols for PRODH-FITC antibodies vary by application type. Based on available data, the following application-specific recommendations can be made:

For Western Blotting (WB):

• Recommended dilution range: 1:2000-1:10000

• Sample types showing positive results: mouse liver tissue, A549 cells, rat liver tissue

• Expected molecular weights: 56 kDa and 66 kDa

For Immunohistochemistry (IHC):

• Recommended dilution range: 1:20-1:200

• Antigen retrieval: Use TE buffer pH 9.0 (primary) or citrate buffer pH 6.0 (alternative)

• Positive tissue samples: human cerebellum, liver cancer, breast cancer, and skeletal muscle tissues

For Immunofluorescence (IF):

• Dilution must be optimized for each specific antibody preparation

• Both cell culture (IF-cc) and paraffin-embedded tissue (IF-p) applications have been validated

• FITC excitation maximum: ~495 nm; emission maximum: ~519 nm

For Flow Cytometry (FACS):

• Several PRODH antibodies have been validated for FACS applications

• Fixation protocol adjustment may be necessary to maintain epitope accessibility

For all applications, it is recommended that researchers titrate the antibody in their specific testing system to obtain optimal results, as outcomes can be sample-dependent .

How can non-specific staining with FITC-labeled PRODH antibodies be minimized?

Non-specific staining is a common challenge with FITC-labeled antibodies, particularly those with higher labeling indices. Several strategies can effectively reduce this issue:

• Optimized antibody dilution: Titrate the antibody to find the optimal concentration that maximizes specific signal while minimizing background. Higher dilutions often reduce non-specific binding .

• Blocking optimization: Extend blocking steps using a combination of serum (matching the host species of the secondary antibody), BSA, and non-fat dry milk to reduce non-specific protein interactions.

• Buffer optimization: Adjust salt concentration and detergent levels in wash buffers to reduce non-specific electrostatic interactions.

• Lower labeling index selection: Choose FITC-conjugated antibodies with moderate labeling indices, as these tend to maintain better specificity despite somewhat lower signal intensity .

• Autofluorescence quenching: Pretreat tissues with Sudan Black B or commercial autofluorescence quenchers, particularly for tissues with high endogenous fluorescence.

• Careful sample preparation: Ensure complete fixation and appropriate antigen retrieval to enhance specific epitope recognition while preserving tissue morphology.

Researchers should systematically test these approaches to determine which combination works best for their specific experimental system and the particular PRODH-FITC antibody being used.

What tissue fixation and antigen retrieval methods are optimal for PRODH-FITC antibody applications?

The choice of fixation and antigen retrieval methods significantly impacts PRODH-FITC antibody performance in tissue samples. Based on available data:

Fixation Recommendations:

• Formalin fixation followed by paraffin embedding is compatible with PRODH antibody detection

• Fixation time should be optimized to maintain antigen integrity while ensuring adequate tissue preservation

• Freshly prepared fixative yields more consistent results than aged solutions

Antigen Retrieval Methods:

• Primary recommendation: TE buffer at pH 9.0

• Alternative approach: Citrate buffer at pH 6.0

• Heat-induced epitope retrieval (pressure cooker or microwave) typically produces better results than enzymatic methods

• Optimization of retrieval duration is essential to balance epitope exposure with tissue integrity

The effectiveness of these methods may vary depending on the specific epitope targeted by the PRODH antibody. Some antibodies targeting amino acids 71-134 may have different optimal retrieval conditions compared to those targeting regions like 441-540 . Researchers should validate these conditions for their specific experimental system and antibody.

How can PRODH-FITC antibodies be utilized in multiplex immunofluorescence studies?

Multiplex immunofluorescence with PRODH-FITC antibodies requires careful planning to avoid spectral overlap and maximize information yield. The following methodological approach is recommended:

• Spectral compatibility planning: FITC (excitation ~495 nm, emission ~519 nm) pairs well with fluorophores like Cy3, Cy5, or Alexa Fluor 594/647 due to minimal spectral overlap.

• Sequential antibody application: For co-localization studies with multiple antibodies, use sequential rather than simultaneous application to reduce cross-reactivity.

• Cross-adsorbed secondary antibodies: When using indirect detection methods alongside FITC-conjugated primaries, employ highly cross-adsorbed secondary antibodies to prevent species cross-reactivity.

• Spectral unmixing: For complex multiplex panels, employ spectral unmixing algorithms during image acquisition or post-processing to separate overlapping fluorescent signals.

• Nuclear counterstaining optimization: When using DAPI or similar nuclear counterstains, adjust concentration to avoid overwhelming the FITC signal (typically in the green channel).

These approaches enable simultaneous detection of PRODH alongside other proteins of interest, facilitating studies of protein co-localization, signaling pathway interactions, and complex tissue architecture analyses. The selection of complementary markers should be guided by the specific research questions and the biological context of PRODH expression.

What are the considerations for using PRODH-FITC antibodies in live-cell imaging?

While FITC-conjugated antibodies can theoretically be used for live-cell imaging, several important considerations must be addressed:

• Membrane permeability: Since PRODH is a mitochondrial protein, membrane permeability remains a significant challenge. Cell-penetrating peptide conjugation or membrane permeabilization techniques may be required.

• Phototoxicity management: FITC is prone to photobleaching and can generate reactive oxygen species upon excitation, potentially damaging live cells. Reducing exposure times, laser power, and employing antifade agents can mitigate these effects.

• Physiological conditions: Maintain physiological pH during imaging as FITC fluorescence is pH-sensitive, with optimal emission at slightly alkaline pH.

• Signal-to-noise optimization: The combination of cellular autofluorescence and potential non-specific binding requires careful optimization of antibody concentration and washing protocols.

• Temporal considerations: FITC-conjugated antibodies may be internalized and degraded over time in live cells, limiting the duration of imaging sessions.

For mitochondrial targets like PRODH, alternative approaches such as expressing fluorescent protein-tagged PRODH or using membrane-permeable mitochondrial dyes in combination with fixed-cell PRODH-FITC antibody staining in parallel experiments may yield more informative results.

How does epitope selection impact PRODH-FITC antibody performance in different applications?

The epitope targeted by PRODH antibodies significantly influences their performance across various applications. Based on available data, several patterns emerge:

The selection of appropriate epitope targets should consider:

• Protein structure accessibility: Epitopes in highly accessible regions typically yield better results in native protein detection methods.

• Conservation across species: For comparative studies, selecting epitopes conserved across species enables consistent detection methodology.

• Post-translational modifications: Avoid epitopes containing potential phosphorylation, glycosylation, or other modification sites that might interfere with antibody binding.

• Domain-specific detection: For studies focusing on specific functional domains of PRODH, epitope selection should target relevant protein regions.

Researchers should align their epitope selection with their specific experimental goals, considering both the biological question and technical requirements of their chosen detection methods.

How can researchers validate the specificity of PRODH-FITC antibodies?

Validating antibody specificity is crucial for reliable research outcomes. For PRODH-FITC antibodies, a multi-faceted validation approach is recommended:

• Knockdown/Knockout validation: The gold standard approach involves comparing antibody signals in wild-type samples versus those with PRODH gene knockdown or knockout. Multiple publications have validated PRODH antibodies using this approach .

• Western blot correlation: Confirm that immunofluorescence patterns correlate with Western blot results showing bands at the expected molecular weights (56 kDa and 66 kDa for PRODH) .

• Peptide competition: Pre-incubate the antibody with the immunizing peptide before application to samples, which should eliminate specific staining.

• Cross-reactivity assessment: Test the antibody on samples from multiple species to confirm the stated species reactivity profile matches experimental results.

• Subcellular localization verification: Confirm that staining patterns match the expected mitochondrial localization of PRODH through co-localization with established mitochondrial markers.

• Correlation with mRNA expression: Compare antibody staining patterns with PRODH mRNA expression data (e.g., from in situ hybridization or public databases) to verify alignment.

This comprehensive validation strategy ensures that observed signals truly represent PRODH protein rather than non-specific binding or technical artifacts.

What factors influence FITC signal stability in fixed samples over time?

The stability of FITC signals in fixed samples is influenced by multiple factors that researchers should consider when designing long-term studies:

• Mounting medium selection: Anti-fade mounting media specifically designed for fluorescence preservation significantly extend FITC signal durability. Media containing p-phenylenediamine or commercial products like ProLong Gold provide superior protection against photobleaching.

• Storage conditions: Samples should be stored at -20°C in the dark. Room temperature storage dramatically accelerates FITC signal decay.

• Slide sealing: Proper sealing of coverslips prevents oxidation and desiccation, which can cause signal degradation.

• Initial signal intensity: Samples with stronger initial fluorescence typically retain detectable signal for longer periods.

• Fixation method impact: The choice of fixative affects signal longevity; paraformaldehyde-fixed samples often maintain fluorescence longer than those fixed with methanol or acetone.

• Exposure history: Minimizing exposure during initial imaging sessions preserves signal for subsequent analysis. Record acquisition settings to enable comparison of images taken at different timepoints.

For critical experiments requiring repeated imaging or long-term storage, researchers should consider preparing duplicate samples or implementing image acquisition strategies that minimize exposure of the entire sample during initial imaging sessions.

How should researchers compare results from different PRODH antibody preparations?

When comparing results obtained with different PRODH antibody preparations, researchers should implement a systematic approach:

• Standardized protocols: Use identical protocols for sample preparation, antibody application, and image acquisition to enable direct comparisons.

• Side-by-side testing: Process samples in parallel rather than sequentially to minimize technical variations.

• Calibration standards: Include identical positive control samples across all antibody tests to establish internal reference points.

• Quantitative analysis: Employ digital image analysis with standardized parameters to objectively compare staining intensity and pattern distribution.

• Epitope consideration: Compare antibodies targeting the same epitope region when possible, as different epitopes may show distinct accessibility in certain applications or sample types.

• FITC-labeling index documentation: For FITC-conjugated antibodies, record the labeling index (if available) to account for sensitivity and specificity differences .

When transitioning between antibody preparations (e.g., switching suppliers or lots), a validation period with parallel testing is strongly recommended to establish correlation and ensure research continuity.