DMXL2 Antibody, FITC conjugated

What is DMXL2 and why is it an important research target?

DMXL2, also known as rabconnectin-3, is a functional regulator of mammalian Notch signaling with WD domains. This protein serves as a scaffold molecule for both Rab3 GEP and GAP on synaptic vesicles, making it crucial for neuronal function. DMXL2 is abundantly expressed in the brain where it is enriched in the synaptic vesicle fraction, with immunofluorescence and immunoelectron microscopy revealing its concentration on synaptic vesicles at synapses . Beyond the brain, DMXL2 plays important roles in multiple organ systems including the heart, kidneys, gonads, and adrenal glands during development .

Research has demonstrated DMXL2's significance in neurobiological mechanisms, particularly in puberty onset in primates and olfactory information transmission . The protein's complex expression pattern - with tissue-specific and developmental stage-dependent variations - makes it an intriguing target for researchers investigating developmental biology, neuroscience, and endocrine function. Given its critical biological roles, antibodies targeting DMXL2 provide valuable tools for elucidating its location, expression patterns, and functional significance in various experimental contexts.

What are the key differences between unconjugated and FITC-conjugated DMXL2 antibodies?

Unconjugated DMXL2 antibodies, such as those described in search results and , require secondary detection methods in applications like Western blotting, immunohistochemistry, and immunoprecipitation. In contrast, FITC-conjugated DMXL2 antibodies have fluorescein isothiocyanate directly attached to the antibody molecule, enabling direct fluorescent detection without the need for secondary antibodies .

The primary advantages of FITC-conjugated antibodies include simplified experimental protocols (elimination of secondary antibody incubation steps), reduced background in fluorescence applications, and compatibility with multicolor immunofluorescence when combined with antibodies conjugated to spectrally distinct fluorophores. The FITC-conjugated DMXL2 antibody (AA 2471-2657) is particularly useful for direct visualization of DMXL2 in fluorescence microscopy applications .

What molecular weight forms of DMXL2 should researchers expect to detect?

Researchers working with DMXL2 antibodies should anticipate detecting multiple molecular weight forms, depending on the tissue source and developmental stage. The calculated molecular weight of full-length DMXL2 is 340 kDa, containing 3036 amino acids . This larger isoform predominates in most tissues, especially in the brain where DMXL2 is abundantly expressed.

When designing experiments to detect DMXL2, researchers should use positive controls from tissues known to express DMXL2 at high levels (e.g., brain tissue, particularly cerebellum) and should be prepared to observe multiple bands. The experimental protocol should be optimized to ensure detection of both high molecular weight species (requiring lower percentage gels and extended transfer times) and smaller isoforms. The appearance of these different isoforms may itself be a valuable experimental endpoint when investigating tissue-specific DMXL2 processing and function.

What are the optimal experimental conditions for immunofluorescence using FITC-conjugated DMXL2 antibodies?

For optimal immunofluorescence using FITC-conjugated DMXL2 antibodies, researchers should implement a carefully controlled protocol that preserves both antigen integrity and fluorophore activity. Begin with appropriate fixation; paraformaldehyde (4%) is generally preferred for FITC applications as it maintains cellular structure while preserving fluorophore activity. For tissue sections, antigen retrieval is critical - use citrate buffer (10 mM, pH 6.0) heated for 7 minutes in a microwave, similar to the protocol used for DMXL2 detection in neural tissues .

The FITC fluorophore is sensitive to photobleaching and has an excitation maximum at approximately 495 nm and emission maximum at 519 nm. Therefore, minimize exposure to light during all handling steps, mount slides with anti-fade mounting medium containing DAPI for nuclear counterstaining, and image using appropriate filter sets. Because DMXL2 shows distinct subcellular localization patterns (e.g., enrichment in synaptic vesicles in neurons), confocal microscopy is recommended for detailed localization studies.

For validation, include positive controls such as Jurkat cells, which have been confirmed to express DMXL2 , and implement negative controls including isotype-matched non-specific IgG at equivalent concentrations to rule out non-specific binding .

How should researchers optimize DMXL2 antibody performance across different tissue types?

Optimizing DMXL2 antibody performance across different tissue types requires careful consideration of tissue-specific expression patterns, fixation methods, and antigen retrieval techniques. Based on the literature, DMXL2 expression varies significantly among tissues, with high expression in brain, heart, kidneys, gonads, and adrenal glands, but lower or no detectable expression in tissues like liver and pancreas at certain developmental stages .

For tissues with high DMXL2 expression (e.g., brain), dilute antibodies more extensively (1:5000-1:10000 for Western blotting) to prevent oversaturation. For tissues with lower expression, use more concentrated antibody solutions (1:2000 for Western blotting) and consider longer incubation times. When examining developmental profiles, note that expression patterns change - DMXL2 is detectable in heart and kidneys at P0 but not in adult animals .

Fixation protocols must be tissue-appropriate. For immunohistochemistry of neural tissues, overnight fixation in Bouin's solution followed by paraffin embedding has proven effective . For gonadal tissue, where strong DMXL2 signal is detected in germ cell cytoplasm, paraformaldehyde fixation may be preferred .

Antigen retrieval is critical for many tissues. For DMXL2 detection in human cerebellum and kidney tissue, use TE buffer at pH 9.0, with citrate buffer (pH 6.0) as an alternative . For neural tissue, microwave heating in 10 mM citrate buffer for 7 minutes has been effective .

Finally, tissue-specific background reduction strategies may be necessary. For highly vascularized tissues, additional blocking of endogenous peroxidases and biotin may be required when using detection systems involving these components.

What controls are essential when using DMXL2 antibodies in experimental protocols?

When utilizing DMXL2 antibodies, especially FITC-conjugated variants, implementing comprehensive controls is essential for ensuring reliable and interpretable results. Five categories of controls should be considered:

• Positive Tissue Controls: Include samples known to express DMXL2 at high levels. Mouse cerebellum tissue, Jurkat cells, and rat brain tissue have been validated for Western blotting . Human cerebellum and kidney tissues are effective for immunohistochemistry .

• Negative Tissue Controls: Include tissues known to express minimal DMXL2, such as adult liver and pancreas at certain developmental stages . This helps establish the specificity of the staining pattern.

• Antibody Controls:

  • Isotype control: Use non-specific rabbit IgG (for polyclonal antibodies) or mouse IgG2b (for monoclonal antibodies) at the same protein concentration as the primary antibody .

  • Antibody titration: Test serial dilutions to determine optimal concentration, as recommended dilutions vary by application (1:50-1:500 for IHC, 1:2000-1:10000 for WB) .

  • For FITC-conjugated antibodies, include an unconjugated isotype control to assess autofluorescence.

• Peptide Competition Controls: Pre-incubate the antibody with its specific immunogen (such as the DMXL2 fusion protein Ag19720) to confirm binding specificity .

• Genetic Controls: When available, include samples from DMXL2 knockout models as the ultimate specificity control. The literature describes Dmxl2 knockout mice that can serve as ideal negative controls .

For internal normalization in quantitative applications, reference proteins should be carefully selected based on the experimental context. For whole hemi-hypothalamic samples, 18S ribosomal RNA shows less variation across developmental stages, while β-actin is more suitable for normalization when comparing anterior and posterior hypothalamic expression .

How can researchers address weak or absent DMXL2 signal in immunofluorescence experiments?

When researchers encounter weak or absent DMXL2 signal in immunofluorescence experiments using FITC-conjugated antibodies, a systematic troubleshooting approach is essential. First, evaluate fixation methods which can significantly impact epitope accessibility. While paraformaldehyde fixation is generally suitable for immunofluorescence, overfixation can mask epitopes. For DMXL2 detection in certain tissues, Bouin's solution fixation followed by paraffin embedding has proven effective .

Antigen retrieval is often critical for DMXL2 detection. If signal is weak, optimize antigen retrieval using either TE buffer at pH 9.0 (primary recommendation) or citrate buffer at pH 6.0 as an alternative . For tissue sections, heat-induced epitope retrieval using microwave heating in 10 mM citrate buffer for 7 minutes may enhance signal .

Antibody concentration and incubation conditions should be carefully optimized. While a dilution range of 1:400-1:1600 is recommended for immunofluorescence applications , tissues with lower DMXL2 expression may require more concentrated antibody solutions. Extending primary antibody incubation to overnight at 4°C rather than shorter incubations at room temperature often improves signal intensity .

For FITC-conjugated antibodies specifically, photobleaching can cause signal loss. Minimize light exposure during all handling steps, use freshly prepared mounting medium with antifade properties, and consider capturing images from unexposed areas of the slide first.

Finally, consider developmental and tissue-specific expression patterns. DMXL2 expression varies significantly across tissues and developmental stages - it's detectable in heart and kidneys at P0 but not in adult animals . Ensure the selected tissue actually expresses DMXL2 by running parallel Western blot analysis to confirm protein presence before concluding that immunofluorescence methodology is problematic.

How should researchers interpret multiple bands or unexpected molecular weights when using DMXL2 antibodies in Western blotting?

When working with brain tissues, two primary bands at 340 kDa and 104 kDa are typically observed in mouse samples . In other tissues, two or three smaller isoforms ranging from 180 to 250 kDa may be detected, with the pattern varying by tissue type . These tissue-specific patterns likely reflect alternative splicing, post-translational modifications, or regulated proteolysis rather than antibody cross-reactivity.

To distinguish between specific isoforms and non-specific binding:

• Compare observed bands with positive controls (e.g., mouse cerebellum, rat brain, or Jurkat cells)

• Conduct peptide competition assays using the specific immunogen

• Compare results across different DMXL2 antibodies targeting distinct epitopes

• When possible, include samples from DMXL2 knockout models as negative controls

For quantitative analyses, researchers should decide a priori which isoform(s) to quantify based on their research question. For studies of total DMXL2 expression, summing the densitometry values of all specific bands may be appropriate. For isoform-specific studies, separate quantification of each band is necessary. In comparative studies across tissues, be aware that the predominant isoform may differ between tissues and developmental stages.

What are the potential sources of false positive or false negative results when using DMXL2 antibodies?

False positive and false negative results with DMXL2 antibodies can arise from multiple sources that researchers must systematically address. For false positives, cross-reactivity represents a significant concern. Some DMXL2 antibodies may recognize structurally similar proteins, particularly other WD-domain containing proteins. To mitigate this risk, validate antibody specificity through peptide competition assays using the specific immunogen (such as DMXL2 fusion protein Ag19720) . Additionally, excessive antibody concentration can increase non-specific binding - adhere to recommended dilutions (1:50-1:500 for IHC, 1:2000-1:10000 for WB) and optimize for each experimental system .

Endogenous enzyme activities can generate false positives in certain detection systems. For IHC applications using HRP-conjugated detection, inadequate blocking of endogenous peroxidase can produce false positive signals. This is particularly relevant in highly vascularized tissues where endogenous peroxidase activity is elevated. Use appropriate peroxidase blocking reagents such as those in the DakoCytomation LSAB+ system .

For false negatives, improper sample preparation represents a primary concern. DMXL2 is a high molecular weight protein (340 kDa) that requires special handling for efficient transfer in Western blotting - use low percentage gels (6-8%), extended transfer times, and specialized buffer systems for large proteins. Inadequate antigen retrieval in fixed tissues frequently causes false negatives; for DMXL2 detection, TE buffer at pH 9.0 is recommended, with citrate buffer (pH 6.0) as an alternative .

Developmental and tissue-specific expression patterns can also lead to misinterpretation. DMXL2 expression changes dramatically during development - it's detectable in heart and kidneys at P0 but not in adult animals . Without knowledge of these patterns, researchers might misinterpret developmental downregulation as technical failure.

For FITC-conjugated antibodies specifically, photobleaching during handling or storage can reduce signal intensity, potentially leading to false negatives. Protect samples from light exposure and use freshly prepared reagents when possible.

How can DMXL2 antibodies be effectively used in developmental studies across different tissue types?

DMXL2 antibodies, particularly FITC-conjugated variants, offer powerful tools for developmental studies due to DMXL2's dynamic expression patterns across different tissues and developmental stages. To effectively implement these antibodies in developmental research, researchers should adopt stage-specific and tissue-specific approaches that account for DMXL2's variable expression patterns.

For studies of neural development, DMXL2 antibodies can trace the protein's expression in the hypothalamus, which plays a critical role in puberty onset . When examining hypothalamic tissues across developmental stages, researchers should normalize gene expression to 18S ribosomal RNA, which shows minimal variation throughout postnatal development . For comparing expression between specific brain regions (e.g., anterior versus posterior hypothalamus), β-actin provides a more suitable reference gene.

In reproductive system development, DMXL2 antibodies reveal dynamic expression patterns in gonads of both sexes. Immunohistochemistry shows strong expression in germ cell cytoplasm and fainter signals in supporting cells (granulosa and Sertoli cells) at P5 and P28 . Researchers should note that Dmxl2 transcript levels remain constant during fetal and early postnatal development but increase markedly with the initiation of spermatogenesis after P5, peaking at P28 when the first elongating spermatids are detected .

For developmental studies of non-neural tissues, researchers should be aware that DMXL2 expression in some organs is transient - it's detectable in heart and kidneys at P0 but not in adult animals . This transient expression pattern suggests developmental stage-specific functions that can be explored through carefully timed immunofluorescence studies.

When designing multi-tissue developmental studies, implement a unified sampling strategy that captures critical developmental milestones (e.g., E18.5, P0, P5, P15, P28, and adult) across all targeted tissues to facilitate direct comparisons . This approach enables correlation of DMXL2 expression changes with specific developmental events across multiple organ systems.

What approaches should be used to investigate DMXL2 interactions with other proteins in synaptic vesicle regulation?

Investigating DMXL2 interactions with other proteins in synaptic vesicle regulation requires sophisticated approaches that can capture both physical associations and functional relationships. DMXL2 (rabconnectin-3) is enriched in synaptic vesicle fractions and may serve as a scaffold molecule for both Rab3 GEP and GAP on synaptic vesicles . To elucidate these interactions, researchers should employ complementary techniques that span from molecular to cellular levels.

Co-immunoprecipitation (Co-IP) represents a foundational approach, with DMXL2 antibodies shown to be effective for immunoprecipitation of the native protein from brain tissue. The recommended protocol uses 0.5-4.0 μg of antibody for 1.0-3.0 mg of total protein lysate . To optimize Co-IP for synaptic vesicle proteins, use detergent conditions that maintain membrane protein interactions - mild detergents like CHAPS or digitonin rather than stronger detergents like Triton X-100. After immunoprecipitation, perform mass spectrometry analysis to identify novel interaction partners beyond known associates like Rab3 GEP and GAP.

For visualization of protein co-localization in neuronal tissues, combine FITC-conjugated DMXL2 antibodies with antibodies against suspected interaction partners labeled with spectrally distinct fluorophores. Confocal microscopy with high-resolution imaging techniques such as Stimulated Emission Depletion (STED) microscopy can resolve co-localization at the synaptic vesicle level.

Functional validation of identified interactions should employ both loss-of-function and gain-of-function approaches. Conditional knockout models targeting DMXL2 specifically in neurons can reveal functional consequences of DMXL2 deletion on putative interaction partners' localization and activity . Complementary approaches using proximity ligation assays (PLA) can confirm direct protein-protein interactions in situ with greater sensitivity than conventional co-localization studies.

For dynamic interaction studies during synaptic vesicle cycling, combine live imaging using FITC-conjugated DMXL2 antibody fragments (Fab) with stimulation protocols that trigger synaptic vesicle exocytosis and endocytosis. This approach can reveal activity-dependent changes in DMXL2 interactions with other synaptic vesicle regulatory proteins.

How can dual immunolabeling with FITC-conjugated DMXL2 antibodies be optimized for co-localization studies?

Optimizing dual immunolabeling with FITC-conjugated DMXL2 antibodies for co-localization studies requires careful consideration of spectral properties, antibody compatibility, and imaging parameters. The FITC fluorophore conjugated to DMXL2 antibodies has excitation/emission maxima at approximately 495/519 nm, which positions it in the green spectrum. For dual labeling, select a second fluorophore with minimal spectral overlap - red fluorophores such as Cy3 (550/570 nm) or far-red fluorophores such as Cy5 (650/670 nm) are ideal partners.

The sequential labeling approach typically yields superior results for DMXL2 co-localization studies. First apply the FITC-conjugated DMXL2 antibody at the optimized dilution (1:400-1:1600) , followed by thorough washing and subsequent application of the second primary antibody. This approach minimizes potential cross-reactivity between detection systems. If the second primary antibody is from the same host species as the DMXL2 antibody, use a monovalent Fab fragment blocking step between applications to prevent cross-binding.

When studying DMXL2 co-localization with synaptic vesicle proteins, tissue preparation becomes particularly critical. For brain tissue, perfusion fixation with 4% paraformaldehyde followed by cryosectioning preserves fine subcellular structures better than paraffin embedding for fluorescence applications. For neuronal cultures, light fixation (2% paraformaldehyde for 15 minutes) maintains synaptic architecture while preserving antigenicity.

For imaging co-localization, confocal microscopy with appropriate narrow bandpass filters is essential to prevent bleed-through between channels. Acquire images sequentially rather than simultaneously to further minimize cross-channel contamination. Include single-labeled controls for each fluorophore to establish proper exposure settings and verify absence of bleed-through.

Quantitative co-localization analysis should employ multiple metrics beyond visual assessment. Calculate Pearson's correlation coefficient, Manders' overlap coefficients, and intensity correlation quotients using image analysis software. For DMXL2 studies, where partial co-localization with other synaptic proteins is expected, object-based co-localization methods may provide more biologically relevant information than pixel-based methods. These approaches identify discrete objects (e.g., synaptic vesicles) in each channel and then determine the percentage of objects that contain both signals.

What considerations are important when using DMXL2 antibodies to study notch signaling regulation?

DMXL2 functions as a regulator of mammalian Notch signaling, making DMXL2 antibodies valuable tools for investigating this critical developmental pathway . When studying DMXL2's role in Notch signaling regulation, researchers must address several important considerations spanning experimental design, tissue selection, and data interpretation.

For co-localization studies between DMXL2 and Notch pathway components, FITC-conjugated DMXL2 antibodies offer advantages when paired with antibodies against Notch receptors (Notch1-4), ligands (Jagged1-2, Delta-like1,3,4), or downstream effectors (RBPJ, Hes/Hey family proteins). Since Notch signaling involves membrane-bound receptors and nuclear translocation of cleaved intracellular domains, subcellular fractionation followed by immunoblotting with DMXL2 antibodies can reveal compartment-specific interactions.

Functional studies require careful consideration of DMXL2's dual roles. Beyond Notch regulation, DMXL2 serves as a scaffold for Rab3 GEP and GAP on synaptic vesicles . To distinguish between these functions, researchers should simultaneously monitor both Notch target gene expression (using qRT-PCR for genes like Hes1, Hes5, and Hey1) and vesicular trafficking markers when manipulating DMXL2 levels.

Genetic approaches provide powerful tools for studying DMXL2-Notch interactions. The available conditional knockout systems for DMXL2, including tissue-specific deletions using Vasa-Cre (germ cells), Amh-Cre (Sertoli cells), and Amhr2-Cre (granulosa cells), enable targeted investigation of DMXL2's role in Notch signaling within specific cellular contexts . These can be combined with Notch reporter systems to directly visualize how DMXL2 deletion affects Notch activity in vivo.

For quantitative analyses of DMXL2's impact on Notch signaling, normalize gene expression appropriately - 18S ribosomal RNA for whole hypothalamic samples and β-actin for comparing expression between anterior and posterior hypothalamus have proven effective .