CD164L2 Human

What is CD164L2 and which protein family does it belong to?

CD164L2 (CD164 sialomucin-like 2 protein) is a human protein belonging to the CD164 family . When investigating this protein, researchers should employ comparative genomics approaches to analyze sequence homology with other CD164 family members. This comparative analysis can provide valuable insights into conserved domains and potential functional similarities. Consider using multiple sequence alignment tools followed by phylogenetic analysis to establish evolutionary relationships within the CD164 family tree.

Where is the CD164L2 gene located in the human genome?

The human CD164L2 gene maps to chromosome 1p36.11 . When studying this genomic region, researchers should consider:

These approaches can reveal regulatory relationships and potential co-expression patterns with neighboring genes that might suggest functional connections or shared biological pathways.

What is the amino acid sequence of human CD164L2?

The amino acid sequence of the recombinant human CD164L2 protein fragment (spanning amino acids 30-141) is: MGSSHHHHHHSSGLVPRGSHM GSGKGARGFGRGALIRLNIWPAVQGACKQLEVCEHCVEGDRARNLSSCMWEQCRPEEPGHCVAQSEVVKEGCSIYNRSEACPAAHHH PTYEPKTVTTGSPPVPEAHSPGFDGAS .

For comprehensive structural characterization, researchers should:

• Note that this sequence includes an N-terminal His-tag (MGSSHHHHHHSSGLVPRGS)

• Perform domain prediction using tools like InterPro or SMART

• Analyze potential post-translational modification sites

• Compare conserved regions with other CD164 family members

• Consider expressing the full-length protein for complete structural studies

How can I use CRISPR/Cas9 systems to study CD164L2 function?

The CD164L2 Double Nickase Plasmid system provides a specific approach for CRISPR/Cas9-mediated gene editing of CD164L2 . These plasmids utilize a D10A mutated Cas9 nuclease paired with target-specific guide RNAs to create highly specific double nicking that mimics double-strand breaks.

For robust experimental design:

• Include comprehensive validation approaches:

  • Genomic PCR and sequencing of the target region

  • Western blot analysis using specific antibodies

  • RT-qPCR for transcript quantification

  • Functional assays based on hypothesized CD164L2 function

• Implement essential controls:

  • Wild-type cells (no transfection)

  • Cells transfected with non-targeting guide RNAs

  • Multiple independent knockout clones

  • Rescue experiments with CD164L2 re-expression

The double nickase approach significantly reduces off-target effects while maintaining high knockout efficiency compared to standard CRISPR systems , making it particularly valuable for studying genes like CD164L2 where specificity is crucial.

What recombinant CD164L2 proteins are available for in vitro studies?

Recombinant human CD164L2 protein fragments are available for research applications. Specifically, a fragment containing amino acids 30-141 expressed in Escherichia coli with >90% purity has been developed for experimental use . This recombinant protein is suitable for applications including SDS-PAGE and mass spectrometry.

When designing experiments with this recombinant protein, researchers should consider:

• Using it as a positive control in Western blots

• Employing it for antibody generation and validation

• Utilizing it as a standard in quantitative assays

• Applying it in protein interaction studies with potential binding partners

How should I design experiments to detect CD164L2 expression?

A comprehensive approach to CD164L2 detection requires multiple methodologies at both RNA and protein levels. While the search results don't provide specific protocols for CD164L2 detection, a methodologically sound approach would include:

• RNA-level detection:

  • Design primers spanning exon junctions to avoid genomic DNA amplification

  • Include positive control tissues/cells based on bioinformatic expression data

  • Validate primer specificity through sequencing of PCR products

  • Consider both standard RT-PCR and quantitative RT-PCR approaches

• Protein-level detection:

  • Use available recombinant CD164L2 as a positive control

  • Include samples from CD164L2 knockout cells as negative controls

  • Optimize antibody concentrations and detection methods

  • Consider both reducing and non-reducing conditions to account for potential disulfide bonds

• Subcellular localization:

  • Employ both biochemical fractionation and imaging approaches

  • Use markers for different cellular compartments as references

  • Consider live-cell imaging for dynamic localization studies

How can I investigate potential functions of CD164L2?

Investigating CD164L2 function requires a multi-modal approach combining computational prediction, genetic manipulation, and functional assays:

• Computational functional prediction:

  • Analyze sequence homology with characterized proteins

  • Examine conserved domains and motifs

  • Explore protein-protein interaction databases for insights

  • Conduct pathway enrichment analysis from co-expression data

• Loss-of-function studies:

  • Implement CRISPR/Cas9-mediated knockout using available tools

  • Compare acute (siRNA) vs. stable (CRISPR) knockdown phenotypes

  • Employ inducible systems for temporal control of expression

• Phenotypic analysis based on CD164 family functions:

  • Assess cell adhesion, migration, and invasive properties

  • Examine effects on cellular signaling pathways

  • Investigate potential roles in differentiation or development

  • Analyze effects on protein trafficking or membrane organization

This systematic approach provides multiple lines of evidence to establish CD164L2 function while minimizing the risk of misattribution due to experimental artifacts.

How might spatial genomics approaches enhance CD164L2 research?

Spatial genomics technologies can provide unique insights into CD164L2 expression and function within tissue context. Based on current spatial genomics methodologies :

• Implement structured batch experimental design:

  • Select tissue cross-sections strategically to maximize information gain

  • Use expected information gain (EIG) approaches for optimal sampling

  • Consider tissue heterogeneity when planning experiments

• Apply spatial transcriptomics to CD164L2 research:

  • Map CD164L2 expression across different tissue regions

  • Correlate with markers of specific cell types or states

  • Identify spatial relationships with potential interaction partners

• Integrate computational approaches:

  • Apply Gaussian Process models as described by spatial genomics researchers

  • Develop predictive models of CD164L2 expression

  • Implement machine learning for pattern recognition in spatial data

What approaches can reveal CD164L2 protein-protein interactions?

Uncovering the CD164L2 interactome is essential for understanding its function in cellular processes. A comprehensive interaction analysis would employ:

• Affinity-based approaches:

  • Use recombinant CD164L2 as bait in pull-down assays

  • Implement co-immunoprecipitation with specific antibodies

  • Include appropriate controls (tag-only, unrelated protein)

  • Validate key interactions through reciprocal pull-downs

• Proximity labeling methods:

  • Generate CD164L2 fusion constructs with BioID, APEX2, or TurboID

  • Optimize labeling conditions for membrane proteins

  • Perform quantitative proteomics to identify labeled proteins

  • Validate hits through orthogonal methods

• Direct binding assays:

  • Express and purify interaction domains separately

  • Perform surface plasmon resonance or microscale thermophoresis

  • Determine binding kinetics and affinity constants

  • Map interaction interfaces through mutational analysis

These complementary approaches provide both discovery and validation pathways to establish the CD164L2 interaction network with high confidence.

How should I interpret contradictory findings in CD164L2 expression studies?

Contradictory findings in CD164L2 expression studies may arise from technical and biological variables. A methodological approach to resolving these contradictions includes:

• Technical assessment:

  • Compare detection methods (antibodies, primers, detection platforms)

  • Evaluate sample preparation protocols (fixation, extraction methods)

  • Consider detection sensitivity and dynamic range limitations

  • Assess statistical approaches and sample sizes

• Biological considerations:

  • Analyze cell type or tissue specificity of expression

  • Examine developmental or conditional regulation

  • Investigate potential isoforms or splice variants

  • Consider post-translational modifications affecting detection

• Resolution strategies:

  • Design experiments specifically addressing contradictions

  • Use multiple independent detection methods

  • Implement genetic validation (e.g., CRISPR knockout controls )

  • Consider collaborative cross-validation between laboratories

This structured approach transforms apparent contradictions into opportunities for deeper understanding of CD164L2 biology.

What controls are essential for validating CD164L2 knockout experiments?

When using CRISPR/Cas9 tools like the CD164L2 Double Nickase Plasmid , comprehensive validation controls are essential:

This multi-level validation approach ensures that observed phenotypes can be confidently attributed to specific CD164L2 disruption rather than technical artifacts or off-target effects.

How can I analyze CD164L2 sequence variations and their potential impact?

Analysis of CD164L2 sequence variations requires a combination of computational prediction and experimental validation:

• Variation identification:

  • Query genomic databases (gnomAD, dbSNP) for known variants

  • Consider targeted sequencing in populations of interest

  • Analyze cancer mutation databases for somatic variants

• Functional prediction:

  • Apply algorithms like SIFT, PolyPhen-2, or CADD

  • Model structural impact using available sequence information

  • Consider evolutionary conservation of affected residues

  • Predict effects on splicing or expression regulation

• Experimental validation:

  • Generate variant constructs through site-directed mutagenesis

  • Compare expression, localization, and stability of variants

  • Assess functional impact through relevant assays

  • Consider knock-in models using CRISPR/Cas9 technology

This integrated approach connects genotypic variation to potential phenotypic consequences, providing insights into CD164L2 structure-function relationships.

How can I address inconsistent results in CD164L2 detection experiments?

Inconsistent CD164L2 detection can stem from multiple factors. A systematic troubleshooting approach includes:

• Reagent validation:

  • Test antibody specificity using recombinant CD164L2 and knockout controls

  • Validate primer specificity through sequencing of PCR products

  • Consider using multiple antibodies targeting different epitopes

• Protocol optimization:

  • Systematically vary experimental conditions (fixation, extraction, detection)

  • Implement positive and negative controls in each experiment

  • Document all protocol modifications thoroughly for reproducibility

• Sample considerations:

  • Assess sample quality and potential degradation

  • Consider heterogeneity within samples

  • Evaluate potential interfering substances

• Quantification approaches:

  • Use appropriate internal controls for normalization

  • Apply consistent quantification methods across experiments

  • Consider absolute quantification when appropriate

By methodically addressing these factors, researchers can develop robust and reproducible protocols for CD164L2 detection.

What are common pitfalls in CD164L2 functional studies and how can they be avoided?

Functional studies of less-characterized proteins like CD164L2 present several challenges:

• Overinterpretation pitfalls:

  • Avoid attributing all observed phenotypes directly to CD164L2

  • Distinguish between primary and secondary effects

  • Consider compensation by related proteins (other CD164 family members)

  • Implementation: Use acute and chronic depletion strategies to identify temporal differences in phenotypes

• Technical artifacts:

  • Non-specific effects of gene editing tools

  • Tag interference with protein function

  • Overexpression artifacts

  • Implementation: Use multiple methodological approaches and appropriate controls

• Context dependency:

  • Cell type-specific functions

  • Condition-dependent roles

  • Implementation: Test hypotheses across multiple cell types and conditions

• Inadequate controls:

  • Missing validation of knockdown/knockout efficiency

  • Lack of rescue experiments

  • Implementation: Follow comprehensive validation protocols as outlined in section 4.2

Addressing these common pitfalls through careful experimental design ensures more reliable and reproducible insights into CD164L2 function.