DEGP6 Antibody

What is DPP6 and what cellular systems express this protein?

DPP6 (Dipeptidyl Peptidase 6) is a transmembrane protein that plays critical roles in neuronal function, particularly in voltage-gated potassium channel regulation. The protein spans from Thr56 to Asp803 with a notable Glu458Val variation in the protein sequence (Accession # AAA35761) . Experimental evidence demonstrates significant DPP6 expression in:

• Human spinal cord tissue (detectable through immunohistochemistry)

• SHSY5Y human neuroblastoma cell lines (confirmed through flow cytometry)

• Central nervous system tissues more broadly

When designing experiments targeting DPP6, researchers should consider these established expression patterns to select appropriate cellular models for their studies.

What detection methods have been validated for DPP6 antibodies?

Based on available scientific data, DPP6 antibodies have been successfully employed in multiple detection platforms:

For optimal results, researchers should determine specific dilutions for each application and laboratory setting, as antibody performance can vary based on experimental conditions.

What controls are essential when validating DPP6 antibody specificity?

Methodologically sound DPP6 antibody experiments require appropriate controls:

• Negative control: Omission of primary antibody while maintaining secondary antibody incubation demonstrates the absence of non-specific secondary antibody binding. This approach has been documented to produce no detectable signal in spinal cord tissue sections .

• Isotype control: For flow cytometry applications, using a control antibody (such as AB-108-C) with the same isotype but no relevant specificity helps distinguish specific from non-specific binding .

• Positive tissue controls: Using tissues with known DPP6 expression (like spinal cord sections) provides validation of antibody performance.

How should researchers optimize immunohistochemical detection of DPP6 in neuronal tissues?

For researchers investigating DPP6 localization in neuronal tissues, the following methodological approach is recommended:

What methodological considerations apply when using flow cytometry for DPP6 detection?

When designing flow cytometry experiments for DPP6 detection, researchers should address these critical factors:

• Cell preparation: For neuroblastoma cell lines like SHSY5Y, maintain viability and membrane integrity during preparation to preserve DPP6 epitopes .

• Staining protocol: Follow established membrane protein staining protocols that minimize internalization of surface proteins during processing .

• Antibody selection: Use DPP6 polyclonal antibody as primary antibody followed by fluorophore-conjugated secondary antibody (APC-conjugated Anti-Goat IgG Secondary Antibody has been validated) .

• Gating strategy: Establish proper gating using:

  • Forward/side scatter to identify intact cells

  • Viability markers to exclude dead cells

  • Control antibody staining to set negative population boundaries

• Analysis: Present results as histograms comparing experimental samples (filled) against isotype controls (open) to clearly demonstrate specific binding .

How can researchers address non-specific binding in DPP6 immunohistochemistry?

When experiencing high background or non-specific staining in DPP6 immunohistochemistry:

• Blocking optimization: Implement more stringent blocking protocols using:

  • Higher concentration of serum (5-10%) from the same species as the secondary antibody

  • Addition of 0.1-0.3% Triton X-100 for improved permeabilization

  • Extended blocking times (1-2 hours at room temperature)

• Antibody dilution: Further titrate antibody concentrations starting from the validated 5 μg/mL concentration . Systematic testing of dilutions may identify optimal signal-to-noise ratio.

• Washing protocol: Implement more rigorous washing steps between antibody applications:

  • Increase washing buffer volume

  • Extend washing times to 10-15 minutes per wash

  • Increase number of washes (minimum 3-5 washes)

• Tissue preparation: Evaluate fixation protocols and post-fixation storage conditions that may affect epitope accessibility.

• Counterstain optimization: Adjust hematoxylin concentration or incubation time to maintain nuclear definition without obscuring DPP6 staining .

What factors influence reproducibility in DPP6 antibody research?

To enhance experimental reproducibility when working with DPP6 antibodies:

• Antibody validation: Confirm antibody lot consistency through:

  • Western blot analysis for molecular weight verification

  • Known positive controls like SHSY5Y neuroblastoma cells

  • Peptide competition assays to confirm specificity

• Protocol standardization: Document detailed protocols including:

  • Precise antibody concentrations (not just dilutions)

  • Incubation times and temperatures

  • Buffer composition and pH

  • Sample preparation methods

• Sample handling: Standardize:

  • Time between collection and fixation

  • Fixation duration and conditions

  • Storage conditions for preserved samples

  • Antigen retrieval methods

• Quantification methods: Implement objective quantification:

  • Digital image analysis with defined parameters

  • Cell counting protocols for flow cytometry

  • Statistical methods appropriate for data distribution

How can researchers integrate DPP6 detection with functional analyses of ion channels?

Given DPP6's role in modulating voltage-gated potassium channels, researchers can implement these integrated approaches:

• Co-localization studies: Combine DPP6 immunostaining with potassium channel subunit detection to examine spatial relationships in neuronal tissues.

• Sequential analysis workflow:

  • Perform electrophysiological recordings in neuronal models

  • Fix and immunostain the same preparations for DPP6 distribution

  • Correlate functional properties with protein expression patterns

• CRISPR-modified systems: Generate DPP6 knockdown or knockout models and assess:

  • Changes in potassium channel kinetics

  • Alterations in neuronal excitability

  • DPP6 antibody staining patterns in modified vs. wild-type cells

• Proximity ligation assays: Investigate protein-protein interactions between DPP6 and channel components using DPP6 antibodies in combination with potassium channel subunit antibodies.

What considerations apply when investigating DPP6 in neurodegenerative disease models?

For researchers examining potential roles of DPP6 in neurological disorders:

• Expression pattern analysis: Compare DPP6 distribution between:

  • Healthy human spinal cord tissue

  • Disease-affected tissues using the validated immunohistochemistry protocol (5 μg/mL, overnight at 4°C)

• Quantitative assessment: Implement:

  • Systematic sampling approaches across tissue sections

  • Unbiased stereological counting methods

  • Digital image analysis with standardized parameters

• Cellular models: Utilize neuroblastoma cell lines like SHSY5Y that have confirmed DPP6 expression to:

  • Model disease conditions (oxidative stress, protein aggregation)

  • Assess changes in DPP6 expression or localization

  • Evaluate potential therapeutic interventions

• Cross-validation approaches: Combine multiple detection techniques:

  • Immunohistochemistry for localization studies

  • Flow cytometry for quantitative expression analysis

  • Western blotting for total protein assessment

  • PCR for transcript level evaluation

How can single-cell techniques incorporate DPP6 antibody detection?

As single-cell analysis advances, researchers can integrate DPP6 antibody detection through:

• Single-cell flow cytometry: Combine DPP6 antibody staining with:

  • Additional neuronal markers

  • Functional indicators

  • Viability assessments

• Imaging mass cytometry: Incorporate metal-conjugated DPP6 antibodies into multiplexed panels for spatial analysis of multiple proteins within tissue sections.

• Patch-seq approaches: Link:

  • Electrophysiological recordings of individual neurons

  • DPP6 immunostaining of the same cells

  • Transcriptional profiling after recording and imaging

• Spatial transcriptomics: Correlate DPP6 protein distribution (via immunostaining) with gene expression patterns in the same tissue sections.

What technological advancements might improve DPP6 antibody research?

Future directions for enhanced DPP6 detection and characterization include:

• Super-resolution microscopy: Apply techniques like STORM or STED to precisely localize DPP6 in relation to potassium channel complexes at the nanoscale level.

• Live-cell imaging approaches: Develop non-fixation dependent methods using:

  • Fluorescently tagged non-blocking DPP6 antibody fragments

  • Genetically encoded tags with minimal functional interference

• Multiplex protein detection: Implement simultaneous detection of DPP6 with channel subunits and regulatory proteins using:

  • Spectrally distinct fluorophores

  • Sequential staining protocols

  • Antibody stripping and reprobing strategies

• Automation of analysis: Develop machine learning algorithms to:

  • Quantify DPP6 expression patterns

  • Identify subtle changes in distribution

  • Correlate expression with functional outcomes

What are the current consensus protocols for DPP6 antibody applications?

Based on available scientific evidence, researchers investigating DPP6 should:

• For immunohistochemistry:

  • Use affinity-purified polyclonal antibodies at 5 μg/mL concentration

  • Implement overnight incubation at 4°C

  • Include appropriate visualization systems and counterstains

  • Always run parallel negative controls without primary antibody

• For flow cytometry:

  • Follow established membrane protein staining protocols

  • Include appropriate isotype controls

  • Utilize fluorophore-conjugated secondary antibodies for detection

  • Present data with clear comparison to control staining

• For all applications:

  • Validate antibody specificity in known positive controls

  • Determine optimal concentrations through systematic titration

  • Document detailed protocols to ensure reproducibility