Dmrt2 Antibody

What is Dmrt2 and why is it significant in research?

Dmrt2 (Doublesex and Mab-3 Related Transcription Factor 2) is a transcription factor belonging to the DMRT family characterized by a conserved DNA-binding motif known as the DM domain. Research has established Dmrt2 as a multifunctional regulator in several critical biological processes:

• Glucose metabolism and insulin sensitivity in adipose tissue

• Neuronal development in the embryonic cortex

• Endochondral bone formation during skeletal development

The significance of Dmrt2 in research lies in its diverse regulatory roles across different tissues and developmental stages. Recent studies have demonstrated that Dmrt2 interacts with FXR (Farnesoid X Receptor) to regulate metabolic pathways, suggesting its potential as a therapeutic target for metabolic disorders . Its expression in developing neurons indicates a role in cortical development , while its presence in chondrocytes suggests involvement in skeletal formation .

What types of Dmrt2 antibodies are available for research?

Researchers have access to multiple types of Dmrt2 antibodies with varying characteristics:

The antibodies vary in their specific applications, with some optimized for Western blotting (WB), immunohistochemistry (IHC), flow cytometry (FC), or multiple techniques . When selecting a Dmrt2 antibody, researchers should consider species reactivity, application compatibility, and validation data provided by manufacturers .

How should Dmrt2 antibodies be validated for Western blot applications?

For robust validation of Dmrt2 antibodies in Western blot applications, implement this comprehensive strategy:

• Positive control tissues/cells:

  • Use tissues with known Dmrt2 expression, such as rib cartilage which shows relatively high Dmrt2 expression

  • Kidney tissue as mentioned in validation data for ABE1364 antibody ("0.25 µg/mL detected DMRT2 in 10 µg of human kidney tissue lysate")

  • Adipose tissue samples from control and insulin-resistant models

• Expression manipulation controls:

  • Compare transfected cells expressing Flag-DMRT2 versus non-transfected controls

  • Include RNA interference samples (e.g., sh-DMRT2 treated cells)

• Technical considerations:

  • Optimize antibody concentration (e.g., 0.25 µg/mL for ABE1364)

  • Use housekeeping proteins like α-tubulin or GAPDH for normalization

  • Run parallel blots with different Dmrt2 antibodies to confirm consistent bands

  • Verify expected molecular weight (varies by species and isoform)

Researchers studying Dmrt2 in insulin resistance successfully validated antibodies by demonstrating clear changes in expression following overexpression or knockdown manipulations .

Recommended tissue samples:

• Adipose tissue:

  • Epididymal fat tissues show clear Dmrt2 expression that is decreased in HFD mice but increased following Dmrt2 overexpression

  • Subcutaneous adipose tissues exhibit similar expression patterns

• Neuronal tissues:

  • Embryonic cortical tissue, particularly the cingulate cortex, where "Dmrt2 is robustly expressed at late developmental stages in post-mitotic neurons and maintained until adulthood"

  • Analysis should consider developmental timepoints (E13.5-E18.5 in mouse models)

• Skeletal/cartilage tissues:

  • Rib cartilage shows high expression of Dmrt2 alongside Sox9 target gene Col2a1

  • Tissues undergoing endochondral bone formation

Optimal fixation methods:

• For tissue sections:

  • 4% paraformaldehyde (PFA) fixation for 10 minutes at 4°C has been successfully used for in situ hybridization of Dmrt2 mRNA

  • Standard formalin fixation followed by paraffin embedding for IHC of adipose tissues

• For cultured cells:

  • Standard fixation protocols suitable for immunofluorescence studies examining co-localization of Dmrt2 with other proteins (such as FXR)

• For protein extraction:

  • RIPA buffer with 1% PMSF for protein extraction from cells or tissues

  • Membrane and Cytosol Protein Extraction Kit for membrane protein isolation

The search results indicate that standard fixation protocols are generally effective for Dmrt2 detection, but optimization may be necessary depending on the specific antibody, tissue type, and research question .

How can I optimize Dmrt2 antibody detection for immunofluorescence?

To optimize Dmrt2 antibody detection for immunofluorescence, follow this methodological approach:

• Antibody dilution optimization:

  • Start with manufacturer's recommended dilutions (e.g., 1:50-1:200 for HPA029297)

  • Perform a dilution series (1:25, 1:50, 1:100, 1:200, 1:400)

  • Test on positive control samples with confirmed Dmrt2 expression

• Essential controls:

  • Positive controls: Samples with overexpressed Dmrt2 (transfected with DMRT2 OE)

  • Negative controls: Samples with knockdown Dmrt2 (transfected with sh-DMRT2)

  • Secondary antibody-only controls to assess background fluorescence

  • No primary antibody controls

• Quality assessment metrics:

  • Signal-to-noise ratio

  • Specificity of subcellular localization (Dmrt2 is a transcription factor, so nuclear localization is expected)

  • Consistency with published localization data (e.g., "The IF staining results showed that DMRT2 protein may collocate with FXR protein")

• Application-specific considerations:

  • For co-localization studies (e.g., with FXR), ensure balanced signal intensity between channels

  • For quantitative analyses, select a concentration that provides a linear response range

  • Optimize blocking conditions (typically 1-5% serum or BSA)

  • Consider antigen retrieval methods if needed

Immunofluorescence has been successfully used to visualize Dmrt2, particularly in studies examining its co-localization with interacting proteins like FXR in adipocytes .

How does Dmrt2 expression change in insulin resistance conditions?

Based on multiple studies, there are significant differences in Dmrt2 expression patterns between normal and insulin-resistant adipose tissue:

• Expression level differences:

  • Consistent downregulation: "DMRT2 in adipose tissues from insulin-resistant subjects" was identified as downregulated through bioinformatics analysis

  • Experimental confirmation: "In epididymal fat tissues, HFD upregulated TNF-α and IL-6 mRNA expression but downregulated DMRT2 mRNA expression"

  • Protein-level evidence: "The levels of DMRT2 were decreased in epididymal fat tissues of HFD mice but partially increased by DMRT2 overexpression"

• Tissue-specific patterns:

  • Similar downregulation observed across multiple adipose depots including subcutaneous and epididymal fat

  • Consistent effect in different experimental models: human tissues, mouse HFD-induced insulin resistance, and in vitro adipocyte models

• Functional consequences:

  • Reduced GLUT4 expression in insulin-resistant tissues/cells with low Dmrt2

  • Decreased p-Akt/Akt ratio in insulin-resistant conditions with low Dmrt2

  • Reduced brown adipocyte marker UCP-1 in tissues with decreased Dmrt2

  • Increased inflammatory markers (TNF-α, IL-6) and macrophage infiltration (F4/80 positive cells)

• Interventional effects:

  • Experimental overexpression of Dmrt2 in insulin-resistant models "partially decreased the mRNA expression levels of TNF-α and IL-6"

  • Dmrt2 overexpression increased glucose uptake, GLUT4 levels, and p-Akt/Akt ratio in insulin-resistant adipocytes

These findings collectively demonstrate that Dmrt2 expression is consistently downregulated in insulin-resistant adipose tissue across multiple models, and this downregulation correlates with increased inflammatory markers and impaired insulin signaling pathways .

What is the mechanistic relationship between Dmrt2 and FXR in metabolic regulation?

The relationship between Dmrt2 and FXR (Farnesoid X Receptor) in metabolic regulation involves direct physical interaction and functional cooperation:

• Physical interaction evidence:

  • Direct binding: "DMRT2 and FXR could interact with each other" as demonstrated by co-immunoprecipitation assays using both exogenous tagged proteins (Flag-DMRT2 and FXR-HA) and endogenous proteins

  • Co-localization: "DMRT2 protein may collocate with FXR protein" as shown by immunofluorescence staining

• Expression relationship:

  • Correlated patterns: Both Dmrt2 and FXR show reduced expression in insulin-resistant conditions

  • Regulatory relationship: "In IR adipocytes, DMRT2 overexpression increased but DMRT2 knockdown decreased FXR protein levels"

• Transcriptional cooperation:

  • Promoter activity: "DMRT2 could increase the FXR transcription activity in GLUT4 transcription"

  • Functional verification: Luciferase assays showed that "DMRT2 overexpression increased the GLUT4 promoter activity" and this effect was blocked by the FXR-specific inhibitor DY268

• Metabolic interdependence:

  • Functional requirement: "FXR knockdown enhanced the insulin resistance and attenuated the effects of DMRT2 overexpression upon 3T3-L1 adipocyte insulin resistance"

  • This demonstrates that Dmrt2's beneficial effects on insulin sensitivity require functional FXR

• Downstream pathways affected:

  • Glucose metabolism: Both factors positively regulate glucose uptake and GLUT4 expression

  • Insulin signaling: Both influence the p-Akt/Akt ratio

  • Inflammation: Both suppress inflammatory cytokine expression

  • Bile acid metabolism: Dmrt2 overexpression affected bile acid metabolism-related genes (increased BSEP and SHP, decreased CYP7A1)

This mechanistic relationship suggests a model where Dmrt2 acts as a transcriptional regulator that enhances FXR expression and activity, with subsequent effects on glucose metabolism, insulin signaling, and inflammation in adipocytes .

How does Dmrt2 influence neuronal development in the embryonic cortex?

Based on recent research, Dmrt2 plays several important roles in neuronal development in the embryonic cortex:

• Spatiotemporal expression pattern:

  • Temporal regulation: "Dmrt2 is robustly expressed at late developmental stages in post-mitotic neurons and maintained until adulthood"

  • Spatial specificity: Particularly expressed in the cingulate cortex

  • Developmental timing: First clearly detected from E14.5 in the cortical plate (CP), potentially present at lower levels earlier (E13.5)

• Effects on neural progenitor cells:

  • Proliferation regulation: "Dmrt2 downregulation triggers the decrease in progenitor cells within the cingulate primordium's ventricular zone"

  • Contrasting effects: "Dmrt2.1 overexpression produces the opposite outcome, increasing the ventricular zone proportion of cells at the expense of generating post-mitotic neurons"

• Impact on neuronal differentiation:

  • Influences the balance between proliferation and differentiation

  • Regulates the transition from progenitor to post-mitotic neuron

• Proposed mechanisms:

  • Two potential models:
    a) Non-cell autonomous role: "Many genes we found misexpressed in Dmrt2 downregulation cells encode for secreted molecules that might impact progenitors"
    b) Low-level direct action: "Dmrt2 expression levels are below the ISH detection threshold" in progenitors but functional at low concentrations

• Sex-specific effects:

  • Differential gene expression: When Dmrt2 was downregulated, researchers "retrieved 2.4 times more DEGs [differentially expressed genes] in female than male comparisons"

  • Sex-differential knockdown efficiency: "In male cells... sh Dmrt2 treatment did not result in a statistically significant reduction of Dmrt2 expression" (15.5% reduction) versus females showing "56.25% reduction"

These findings establish Dmrt2 as an important transcriptional regulator in cortical development, influencing the balance between progenitor proliferation and neuronal differentiation, with potentially sex-specific effects that warrant further investigation .

What role does Dmrt2 play in endochondral bone formation?

According to research findings, Dmrt2 plays a significant role in endochondral bone formation with the following specific functions:

• Expression pattern in cartilage:

  • Tissue-specific expression: "Relatively high expression of Dmrt2, as well as the Sox9 target gene Col2a1, in rib cartilage"

  • Temporal regulation: "Dmrt2 expression increased during in vitro chondrocyte differentiation of ATDC5 cells in the presence of insulin–transferrin–selenium (ITS)"

• Functional role in endochondral ossification:

  • Dmrt2 "promotes transition of endochondral bone formation"

  • Specifically involved in the "coordinated transition from a proliferating to a hypertrophic stage" of chondrocytes

  • This transition is described as "critical to advance skeletal development"

• Chondrocyte specificity:

  • Cell-type localization: "Dmrt2 is expressed in chondrocytes during endochondral bone formation"

  • This expression pattern aligns with its functional role in chondrocyte differentiation

The relationship with Sox9 and Col2a1 (a major cartilage matrix protein) suggests Dmrt2 may be part of a transcriptional network that controls cartilage development and subsequent bone formation . While the complete molecular mechanisms remain to be fully elucidated, the data indicates that Dmrt2 functions as a transcription factor regulating genes involved in chondrocyte differentiation during the endochondral ossification process.

Why might I see inconsistent results when using Dmrt2 antibodies in different tissue types?

Inconsistent results when using Dmrt2 antibodies across different tissue types could stem from several factors:

• Isoform-specific expression:

  • Alternative transcripts: "The Dmrt2 locus has three alternative transcript variants" (Dmrt2.1, Dmrt2.2, and Dmrt2.3)

  • Structural differences: "Dmrt2.2 and Dmrt2.3 retain exon 4, which contains an alternative stop codon"

  • Domain variation: "Dmrt2.3 mRNA will be transcribed from a cryptic transcription start site in exon 2... This protein product will lack the DM domain"

  • Antibodies targeting regions specific to certain isoforms may not detect all Dmrt2 variants

• Tissue-specific expression levels:

  • Variable expression: High in rib cartilage, detectable in adipose tissue, developmentally regulated in neuronal tissue

  • Detection challenges: "Transcription factors often function at very low concentrations"

  • Technique limitations: "The fact that we detect expression with RT-qPCR at E13.5 before we can observe any expression in the cingulate primordium through ISH... opens the possibility that Dmrt2 is not detected in the VZ at any time point with this technique"

• Sex-specific expression differences:

  • Differential expression: The data notes sex-specific differences in Dmrt2 expression and function

  • Variable knockdown efficiency: "In male cells... sh Dmrt2 treatment did not result in a statistically significant reduction" versus significant reduction in females

• Protein interactions affecting epitope access:

  • Protein complexes: Dmrt2 interacts with FXR, which could mask antibody epitopes in tissues where this interaction occurs

  • Context-dependent protein associations could affect antibody accessibility

• Limited validation across tissue types:

  • Most antibodies are validated in a limited set of tissues/applications

  • For example, ABE1364 was validated in "human kidney tissue lysate" but may perform differently in other tissues

To address these inconsistencies, researchers should perform comprehensive validation in each tissue type of interest, including positive and negative controls, and consider using multiple antibodies targeting different Dmrt2 epitopes .

How can I address nonspecific binding when using Dmrt2 antibodies?

To address nonspecific binding when using Dmrt2 antibodies, implement these methodological approaches:

• Optimize blocking conditions:

  • Use appropriate blocking buffers containing proteins that reduce nonspecific binding sites

  • Consider tissue-specific blocking agents (e.g., normal serum from the same species as the secondary antibody)

  • Extend blocking time to ensure complete saturation of nonspecific binding sites

• Validate antibody specificity:

  • Use genetic controls: Compare samples with Dmrt2 overexpression vs. knockdown

  • Effective control strategy: "DMRT2 overexpression or knockdown was achieved in adipocytes by transducing DMRT2-overexpressing vector (DMRT2 OE) or small interference RNA for DMRT2 (sh-DMRT2)"

  • These manipulations should produce clear differences in protein detection, confirming specificity

• Optimize antibody concentration:

  • Perform titration experiments: Determine the optimal antibody concentration

  • Follow manufacturer recommendations: For Sigma-Aldrich HPA029297, the recommended dilution range for IHC is "1:50-1:200"

  • Application-specific optimization: For Western blotting, 0.25 µg/mL was effective for the ABE1364 antibody

• Include comprehensive controls:

  • No primary antibody controls to assess secondary antibody nonspecific binding

  • Isotype controls (especially for monoclonal antibodies)

  • Pre-absorption controls with blocking peptides if available

• Optimize washing protocols:

  • Increase washing duration and/or number of washes

  • Use appropriate detergent concentration in wash buffers

  • Consider different detergent types based on the application

• Address cross-reactivity issues:

  • Test antibodies on samples from relevant species

  • Verify specificity for your experimental model

  • Many Dmrt2 antibodies have specified reactivity (e.g., "species reactivity: human, rat" for ABE1364)

• Application-specific considerations:

  • For Western blotting: Use higher percentage SDS-PAGE gels to better separate proteins of similar molecular weight

  • For IHC/IF: Optimize antigen retrieval methods for the specific tissue type

  • For co-IP experiments: Use more stringent washing conditions

• Select appropriate antibody formats:

  • Consider using affinity-purified antibodies like those mentioned in the search results: "affinity isolated antibody" (ABE1364, HPA029297)

  • For challenging applications, monoclonal antibodies may provide higher specificity

By systematically implementing these approaches, researchers can minimize nonspecific binding and improve the signal-to-noise ratio when using Dmrt2 antibodies across different experimental applications .

How can I determine if sex-specific differences affect Dmrt2 detection?

Based on research findings, there are important sex-specific differences that may affect Dmrt2 detection. Here's a methodological approach to determine and account for these differences:

• Experimental design considerations:

  • Always analyze males and females separately

  • Include sufficient sample sizes for each sex to enable statistical comparisons

  • Control for hormonal status in adult animals (e.g., estrous cycle stage in females)

• Evidence of sex-specific differences:

  • Differential gene expression: "We retrieved 2.4 times more DEGs in female than male comparisons"

  • Different knockdown efficiency: "In male cells, we observed that the sh Dmrt2 treatment did not result in a statistically significant reduction of Dmrt2 expression compared to the mock treatment (15.5% reduction; p-adj = 0.4524)" while "females show a 56.25% reduction (p-adj = 0.0216)"

• Multi-method detection approach:

  • Use multiple detection techniques for cross-validation:

    • RT-qPCR for mRNA detection

    • Western blotting for protein detection

    • Immunohistochemistry/immunofluorescence for spatial analysis

  • Compare results between methods to identify discrepancies that might be sex-specific

• Isoform-specific analysis:

  • Analyze all known Dmrt2 isoforms separately

  • Design primers/probes specific to each variant: "Dmrt2.1, Dmrt2.2, and Dmrt2.3"

  • Consider that different isoforms may be differentially expressed between sexes

• Antibody validation protocol for sex differences:

  • Test antibodies on male and female samples in parallel

  • Include positive controls (overexpression) and negative controls (knockdown) for both sexes

  • Verify antibody specificity in the specific tissue of interest from both sexes

• Statistical approaches:

  • Include sex as a variable in statistical models

  • Test for sex-by-treatment interactions

  • When pooling data, verify that patterns are consistent across sexes