DLX5 Antibody

What is DLX5 and why is it an important research target?

DLX5 belongs to the DLX family of homeobox transcription factors that control critical developmental processes. It functions as an immediate early BMP-responsive transcriptional activator essential for osteoblast differentiation and plays a significant role in controlling differentiation of GABAergic neurons in the subpallium . With a molecular weight of approximately 31.5-35 kDa, DLX5 represents an important target for developmental biology, neuroscience, and bone formation studies . DLX5 may also be known by alternative names including SHFM1D (Split Hand/Foot Malformation 1D), homeobox protein DLX-5, and distal-less homeo box 5 .

What should researchers consider when selecting a DLX5 antibody for their experiments?

When selecting a DLX5 antibody, researchers should evaluate:

The wide availability of DLX5 antibodies (261 products across 25 suppliers) necessitates careful selection based on specific experimental requirements .

What cellular localization pattern should be expected when using DLX5 antibodies?

When using DLX5 antibodies, researchers should expect nuclear localization with some cytoplasmic staining. As a transcription factor, DLX5 primarily functions in the nucleus, but may be detected in the cytoplasm depending on cellular state and fixation methods. In human mesenchymal stem cells differentiated into osteocytes, DLX5 has been detected in both nuclei and cytoplasm using immunofluorescence techniques . This dual localization pattern is consistent with the protein's role as a transcription factor that translocates between cellular compartments in response to signaling events.

What are the optimal dilutions and conditions for using DLX5 antibodies in different applications?

Optimal conditions vary by application, antibody source, and experimental system:

These recommendations should serve as starting points, with optimization necessary for each unique experimental system. Titration experiments are strongly recommended to determine optimal signal-to-noise ratios .

What sample preparation techniques are recommended for optimal DLX5 detection?

For optimal DLX5 detection across different applications:

• Western Blot Samples:

  • For cell lines, A549, A375, and U-251 cells have shown positive detection

  • Include phosphatase inhibitors to preserve phosphorylation states at Ser34 and Ser217

  • Use nuclear extraction protocols to enrich for nuclear proteins

• Immunohistochemistry:

  • Human thymus tissue has been validated for DLX5 detection

  • Perform antigen retrieval with TE buffer pH 9.0 (preferred) or alternatively citrate buffer pH 6.0

  • Optimize fixation duration to prevent epitope masking

• Immunofluorescence:

  • For mesenchymal stem cells, fix by immersion fixation

  • Use appropriate secondary antibodies (e.g., NorthernLights™ 557-conjugated for sheep primaries)

  • Counterstain with DAPI to visualize nuclear localization

• General Storage Considerations:

  • Store samples at -20°C to -70°C

  • Avoid repeated freeze-thaw cycles

  • For short-term storage (1 month), 2-8°C under sterile conditions is acceptable

How can researchers troubleshoot non-specific binding or weak signals when using DLX5 antibodies?

When encountering problems with DLX5 antibody performance:

If problems persist, validation using genetic approaches (siRNA knockdown or CRISPR knockout) can confirm specificity .

How can DLX5 antibodies be used to study osteoblast differentiation and bone development?

DLX5 antibodies are valuable tools for studying osteogenic differentiation:

• Developmental Timeline Analysis:

  • Track DLX5 expression changes during mesenchymal stem cell differentiation into osteocytes

  • Correlate with other osteogenic markers to establish differentiation checkpoints

• BMP Signaling Response:

  • Monitor DLX5 levels after BMP treatment to assess immediate early response

  • Compare phosphorylated versus total DLX5 to measure activation state

• Biomaterial Interaction Studies:

  • Evaluate DLX5 expression in cells cultured on different substrates or hydrogels

  • Assess how mechanical properties of hydrogels drive bone formation through DLX5 pathways

• Co-localization Studies:

  • Examine nuclear translocation kinetics using immunofluorescence

  • Analyze co-localization with other transcription factors involved in osteogenesis

• Functional Assessment:

  • Correlate DLX5 expression levels with downstream target gene activation

  • Use in combination with reporter assays to measure transcriptional activity

These approaches have contributed to understanding how hydrogel mechanics influence bone formation by mesenchymal stromal cell spheroids, as demonstrated in recent publications .

What methodologies are recommended for investigating DLX5's role in neuronal development?

To study DLX5's function in neuronal development:

• GABAergic Interneuron Differentiation:

  • Use DLX5 antibodies to monitor expression in the subpallium during development

  • Track DLX5-positive cells during migration and differentiation stages

• Co-expression Analysis:

  • Perform double immunostaining with other DLX family members (DLX1, DLX2, DLX6)

  • Identify temporal and spatial patterns of expression in neural tissues

• Genetic Manipulation Studies:

  • Validate knockdown or overexpression efficiency using DLX5 antibodies

  • Correlate DLX5 levels with neuronal phenotypes and electrophysiological properties

• Signaling Pathway Analysis:

  • Examine how different signaling pathways regulate DLX5 expression in neuronal precursors

  • Study post-translational modifications of DLX5 during neuronal differentiation

• Chromatin Immunoprecipitation:

  • Identify genomic binding sites of DLX5 in neural tissues

  • Compare binding profiles across developmental stages

These approaches can provide insights into DLX5's control of GABAergic neuron differentiation and interneuron development .

How can researchers study protein-protein interactions involving DLX5?

To investigate DLX5's interactions with other proteins:

• Co-immunoprecipitation (Co-IP):

  • Use DLX5 antibodies to pull down protein complexes

  • Identify interaction partners like MSX-2 through western blot or mass spectrometry

  • Both rabbit polyclonal and monoclonal antibodies have been validated for IP applications

• Proximity Ligation Assay (PLA):

  • Visualize protein interactions in situ using primary antibodies against DLX5 and potential partners

  • Quantify interaction frequency in different cellular compartments

• Chromatin Immunoprecipitation (ChIP):

  • Map genomic binding sites of DLX5 using validated antibodies

  • Perform sequential ChIP to identify co-occupancy with other transcription factors

• Functional Interaction Studies:

  • Use reporter assays to measure how DLX5 interactions affect transcriptional activity

  • Compare wildtype versus mutant DLX5 to identify interaction domains

• Bimolecular Fluorescence Complementation:

  • Directly visualize interactions in living cells using fluorescent protein fragments

These methods can elucidate how DLX5 forms homodimers and heterodimers with proteins like MSX-2, as mentioned in the search results .

What controls are essential when using DLX5 antibodies in research applications?

When using DLX5 antibodies, include these critical controls:

Proper controls ensure research validity and reproducibility, particularly when studying a transcription factor like DLX5 that shares homology with other family members.

How should researchers optimize storage and handling of DLX5 antibodies to maintain performance?

To preserve DLX5 antibody functionality:

• Long-term Storage:

  • Store at -20°C to -70°C as supplied

  • Many DLX5 antibodies are formulated with 50% glycerol and 0.02% sodium azide for stability

  • Avoid repeated freeze-thaw cycles

• Short-term Storage:

  • Reconstituted antibodies can be stored at 2-8°C for up to 1 month under sterile conditions

  • For longer storage (up to 6 months), keep at -20°C to -70°C

• Working Solutions:

  • Prepare fresh working dilutions

  • For antibodies without BSA, adding BSA (0.1%) may improve stability

  • Return stock solutions to recommended storage conditions immediately after use

• Shipping and Receipt:

  • Verify cold chain maintenance during shipping

  • Inspect for proper condition upon arrival

  • Allow products to equilibrate before opening

• Documentation:

  • Record lot numbers, receipt dates, and reconstitution information

  • Track performance across experiments to identify potential degradation

Following these guidelines will help maintain antibody performance and experimental reproducibility.

How are DLX5 antibodies being utilized in advanced single-cell analysis techniques?

DLX5 antibodies are increasingly being incorporated into single-cell methodologies:

• Single-cell Immunostaining:

  • Track heterogeneity in DLX5 expression across individual cells within populations

  • Combine with other markers to identify distinct cellular subtypes during differentiation

• Mass Cytometry (CyTOF):

  • Metal-conjugated DLX5 antibodies enable multi-parameter analysis at single-cell resolution

  • Quantify expression levels alongside dozens of other proteins simultaneously

• Spatial Transcriptomics Integration:

  • Correlate DLX5 protein localization with spatial gene expression data

  • Map developmental gradients with cellular resolution

• Lineage Tracing:

  • Track DLX5-expressing cells through development or disease progression

  • Identify cell fate decisions influenced by DLX5 expression levels

• Single-cell Proteomics:

  • Quantify DLX5 levels and post-translational modifications in individual cells

  • Correlate with functional outcomes at single-cell resolution

These emerging techniques extend the utility of DLX5 antibodies beyond traditional bulk analyses, revealing cellular heterogeneity and regulatory mechanisms with unprecedented resolution.

What methodological considerations are important when studying DLX5 phosphorylation and its impact on transcriptional activity?

To investigate DLX5 phosphorylation and its functional consequences:

• Phosphorylation Site Analysis:

  • Focus on known phosphorylation sites (Ser34 and Ser217) that increase transcriptional activity

  • Use phospho-specific antibodies if available, or general phospho-serine antibodies with IP

• Kinase Identification:

  • Design experiments to identify kinases responsible for DLX5 phosphorylation

  • Use kinase inhibitors to examine effects on DLX5 activity and localization

• Phosphorylation Dynamics:

  • Assess temporal changes in phosphorylation status following stimulation (e.g., BMP signaling)

  • Correlate with nuclear translocation and transcriptional activation

• Mutational Analysis:

  • Generate phospho-null (S→A) and phospho-mimetic (S→E/D) mutants

  • Compare transcriptional activity using reporter assays

• Functional Correlation:

  • Link phosphorylation states to biological outcomes in development or pathology

  • Examine phosphorylation patterns in different cellular contexts

These approaches can elucidate how post-translational modifications regulate DLX5's role as a transcriptional activator in bone formation and neuronal development.