DGCR6L Antibody

What is DGCR6L and why is it significant in DiGeorge syndrome research?

DGCR6L is a protein encoded by one of two functional genes resulting from a duplication at the DGCR6 locus. The protein shares homology with the Drosophila gonadal protein and with human laminin gamma-1 chain, which functions in cell attachment and migration . This gene is located in chromosome 22q11, a region implicated in DiGeorge syndrome, which is part of the broader CATCH 22 syndrome complex .

The significance of DGCR6L in DiGeorge syndrome research stems from its location within the commonly deleted region in patients with velo-cardio-facial syndrome/DiGeorge syndrome (VCFS/DGS) . Research suggests it may play a role in neural crest cell migration into the third and fourth pharyngeal pouches, which is critical for understanding the developmental abnormalities associated with DiGeorge syndrome .

How can researchers distinguish between DGCR6 and DGCR6L in experimental systems?

Distinguishing between DGCR6 and DGCR6L requires exploiting the single nucleotide differences between them. According to sequence analysis:

• There are 32 single nucleotide differences between the cDNA sequences of DGCR6 and DGCR6L .

• One reliable distinction is a C at position 167 of DGCR6 and a T at position 168 of DGCR6L in the cDNA sequence, corresponding to a PvuII restriction site within DGCR6 but not in DGCR6L .

Methodology for differentiation:

• Generate a PCR product spanning the region containing these differences (253 bp product using primers between exon 1 and exon 2)

• Perform restriction digestion with PvuII

• The presence of both digested and undigested products indicates the presence of both genes

• Complete digestion indicates only DGCR6 is present

What applications are DGCR6L antibodies validated for in research settings?

DGCR6L antibodies have been validated for multiple applications in research settings:

These antibodies have been tested against human samples, with some also validated for mouse reactivity .

What are the optimal storage and handling conditions for DGCR6L antibodies?

For optimal antibody performance in DGCR6L detection:

• Store antibodies at -20°C and avoid freeze/thaw cycles to maintain activity .

• Many DGCR6L antibodies are supplied in buffers containing PBS with 0.05% NaN3 and 40% Glycerol at pH 7.4 .

• When working with the antibody, aliquot to minimize freeze/thaw cycles.

• Follow manufacturer-recommended dilution factors: 1:1000-1:5000 for Western blot, 1:50-1:300 for IHC, and 1:5000-1:10000 for ELISA applications .

How can researchers effectively use DGCR6L antibodies to study the evolutionary conservation of this protein?

The DGCR6/DGCR6L gene duplication presents a unique opportunity to study evolutionary conservation:

• Research indicates the duplication is at least 12 million years old and may date back to before the divergence of Catarrhines from Platyrrhines (35 million years ago) .

• Methodology for evolutionary studies:

  • Utilize FISH mapping techniques across different ape species to track chromosomal location .

  • Employ PCR amplification of genomic DNA from primate species using primers DGCR6-6F and DGCR6-4R .

  • Analyze sequence conservation through restriction enzyme analysis (e.g., PvuII digestion).

  • Compare expression patterns across species using RT-PCR followed by restriction analysis.

This approach can reveal selective evolutionary pressure toward the functional maintenance of both DGCR6 and DGCR6L paralogs, providing insights into their biological importance .

What methodological considerations should be addressed when using DGCR6L antibodies to study neural crest cell migration?

When investigating neural crest cell migration with DGCR6L antibodies:

• Choose appropriate experimental models: Since DGCR6L may play a role in neural crest cell migration into pharyngeal pouches , consider using:

  • Primary neural crest cell cultures

  • Embryonic tissue sections at developmental stages when migration occurs

  • Cell lines that model neural crest behavior

• Technical approach:

  • Use immunofluorescence with DGCR6L antibodies (dilution 1:50-1:300) along with neural crest markers .

  • Employ time-lapse imaging of labeled cells to track migration patterns.

  • Consider co-staining with laminin gamma-1 antibodies to investigate interaction with DGCR6L in migration .

  • Apply appropriate fixation protocols: 4% paraformaldehyde is generally suitable for maintaining cell morphology during migration studies.

• Controls should include:

  • DGCR6L-knockout or knockdown samples

  • Comparison with DGCR6 expression patterns

  • Analysis in DiGeorge syndrome patient-derived cells versus controls

What are the technical approaches to differentiate DGCR6 and DGCR6L expression in VCFS/DGS patient samples?

Differentiating DGCR6 and DGCR6L expression in patient samples requires specialized techniques:

• Genetic analysis approach:

  • PCR amplification of genomic DNA using primers that span regions with known sequence differences .

  • Restriction enzyme digestion (e.g., PvuII) to distinguish between the genes based on sequence polymorphisms .

  • Quantitative real-time PCR with gene-specific primers designed around unique sequence regions.

• Protein detection approach:

  • Western blot analysis using antibodies that can distinguish between the proteins (if available).

  • The proteins have a calculated MW of 25 kDa but may show slight differences in migration patterns .

  • Use patient samples with known 22q11 deletions as controls, as they contain a single copy of each gene .

• Expression analysis in patient tissues:

  • Design RT-PCR primers that amplify across regions containing single nucleotide differences.

  • Perform restriction digestion of the RT-PCR products to distinguish expression from each gene .

  • Use this approach to compare expression patterns in different tissues of VCFS/DGS patients.

How can researchers optimize the detection of DGCR6L protein interactions with other cellular components?

To investigate DGCR6L protein interactions:

• Co-immunoprecipitation (Co-IP) approach:

  • Use DGCR6L antibodies for immunoprecipitation from cell or tissue lysates.

  • Validate antibody efficiency for IP before proceeding.

  • Analyze precipitated complexes by mass spectrometry to identify interaction partners.

  • Confirm interactions by reciprocal Co-IP and Western blotting.

• Proximity ligation assay (PLA):

  • Utilize DGCR6L antibodies in combination with antibodies against suspected interaction partners.

  • This technique allows visualization of protein-protein interactions in situ with high specificity.

  • Particularly useful for investigating the proposed interaction between DGCR6L and laminin gamma-1 .

• Immunofluorescence co-localization:

  • Use ICC/IF applications of DGCR6L antibodies combined with markers for cellular structures.

  • Analyze co-localization using confocal microscopy and appropriate quantification software.

  • Particularly valuable for examining DGCR6L localization during neural crest cell migration.

What are the critical validation steps for DGCR6L antibodies before experimental use?

Before using DGCR6L antibodies in critical experiments, researchers should:

• Perform specificity testing:

  • Western blot analysis using positive and negative control samples

  • Include DGCR6L overexpression and knockdown controls

  • Test cross-reactivity with DGCR6 due to high sequence homology (97% identity)

• Validate for specific applications:

  • For WB: Confirm the antibody detects a band at the expected molecular weight (25 kDa)

  • For ICC/IF: Verify appropriate cellular localization pattern

  • For IHC: Test on known positive and negative tissue sections with appropriate controls

• Cross-reference with multiple antibodies:

  • When possible, compare results using antibodies from different sources

  • Consider antibodies targeting different epitopes within DGCR6L

  • Compare polyclonal and monoclonal antibodies if available

What technical challenges might researchers encounter when studying DGCR6L in the context of the DiGeorge critical region?

Researchers studying DGCR6L in the DiGeorge critical region face several technical challenges:

• Genomic complexity challenges:

  • The gene is located within low copy repeats (sc11.1b) which can complicate genetic and genomic analyses

  • The presence of a full-length HERV-K provirus in the proximal copy (sc11.1a) must be considered in genomic studies

  • The high sequence homology between DGCR6 and DGCR6L (97%) makes specific targeting difficult

• Sample-related challenges:

  • VCFS/DGS patients typically have hemizygous deletions, resulting in a single copy of each gene

  • 97.4% of patients maintain the C/T nucleotide difference between DGCR6/DGCR6L, but ~2.6% do not, which can confound analyses

  • Limited availability of patient samples with defined genetic characteristics

• Expression analysis challenges:

  • Both genes are widely expressed in fetal and adult tissues, making functional differentiation complex

  • Current antibodies may not clearly distinguish between the two highly similar proteins

  • Need for careful primer design and validation in expression studies

How might DGCR6L antibodies be utilized in emerging single-cell analysis techniques?

DGCR6L antibodies could be integrated into emerging single-cell technologies:

• Single-cell proteomics applications:

  • Incorporation into mass cytometry (CyTOF) panels to correlate DGCR6L expression with cell lineage markers

  • Use in microfluidic single-cell Western blotting to assess protein expression heterogeneity

  • Application in spatial proteomics to map DGCR6L localization within tissue architecture

• Multiomics approaches:

  • Combine with single-cell RNA-seq to correlate protein and mRNA expression levels

  • CITE-seq (Cellular Indexing of Transcriptomes and Epitopes by Sequencing) to simultaneously profile surface proteins and transcriptomes

  • Spatial transcriptomics combined with immunofluorescence to map both RNA and protein expression

• Live cell applications:

  • Development of DGCR6L antibody fragments for live cell imaging

  • Integration with optogenetic approaches to study dynamic protein interactions

  • Combination with super-resolution microscopy techniques for detailed localization studies

What are potential research applications for studying DGCR6L in developmental contexts beyond DiGeorge syndrome?

Based on its homology and expression patterns, DGCR6L research has potential applications in:

• Neural development studies:

  • Investigation of neural crest cell guidance mechanisms beyond pharyngeal arches

  • Potential role in neuronal migration and circuit formation

  • Possible functions in glial cell development and myelin formation

• Cell adhesion and migration research:

  • Based on homology with laminin gamma-1 chain, exploration of roles in extracellular matrix interactions

  • Investigation of potential functions in epithelial-mesenchymal transitions

  • Study of possible roles in cancer cell migration and invasion

• Evolutionary developmental biology:

  • Comparative analysis across species to understand functional conservation

  • Investigation of the evolutionary pressure that maintained both DGCR6 and DGCR6L paralogs

  • Study of the impact of the human-specific HERV-K provirus integration near DGCR6 on gene regulation

What are common troubleshooting strategies when DGCR6L antibodies fail to produce expected results?

When encountering issues with DGCR6L antibody performance:

• Western blot troubleshooting:

  • Adjust antibody concentration (try dilutions from 1:1000 to 1:5000)

  • Optimize protein loading amount (25-50 μg total protein)

  • Try different blocking agents to reduce background

  • Consider different detection systems (chemiluminescence vs. fluorescent)

  • Test samples under both reducing and non-reducing conditions

• Immunohistochemistry/Immunocytochemistry troubleshooting:

  • Adjust antibody dilution (1:50 to 1:300 range)

  • Test different fixation methods (4% PFA, methanol, acetone)

  • Try different antigen retrieval methods (heat-induced vs. enzymatic)

  • Extend primary antibody incubation time (overnight at 4°C)

  • Use signal amplification systems for low-abundance targets

• General troubleshooting considerations:

  • Validate antibody on positive control samples with known DGCR6L expression

  • Consider antibody lot variation and request technical support from manufacturers

  • Test alternative antibodies targeting different epitopes of DGCR6L

  • Check sample handling and storage conditions