DEDD2 Antibody

What is the optimal protocol for detecting DEDD2 in immunofluorescence studies?

For optimal detection:

• Fix cells with 4% paraformaldehyde for 15 minutes at room temperature

• Permeabilize with 0.1% Triton X-100 for 10 minutes

• Block with 5% normal serum for 1 hour

• Incubate with anti-DEDD2 antibody (1:200-1:500 dilution) overnight at 4°C

• Use fluorochrome-conjugated secondary antibodies specific to your primary antibody's host species

To co-localize DEDD2 with intermediate filaments, consider dual staining with antibodies against keratin 8 or 18, as DEDD2 has been shown to colocalize with these cytoskeletal elements during apoptosis .

How can I differentiate between DEDD and DEDD2 in my experimental system?

Differentiating between DEDD and DEDD2 requires careful antibody selection and validation:

• Use antibodies targeting unique epitopes: DEDD2-specific antibodies should target regions that differ from DEDD, particularly outside the conserved death effector domain (DED). Commercial DEDD2 antibodies often use recombinant fusion proteins containing amino acids 1-240 of human DEDD2 (NP_579874.1) as antigens .

• Perform siRNA knockdown validation: Transfect cells with siRNAs specific to either DEDD or DEDD2 and confirm antibody specificity by Western blotting.

• Consider expression pattern differences: DEDD and DEDD2 show distinct expression kinetics in response to stimuli. For example, in DCs treated with LPS or TNF-α, DEDD2 expression increases significantly at 8 hours but returns to baseline by 48 hours, while induced DEDD expression is sustained .

• Molecular weight distinction: DEDD exists in multiple forms (37 kD unmodified, 44 kD monoubiquitinated, and 54 kD diubiquitinated) , which can help differentiate it from DEDD2 on Western blots.

What are the recommended antibody dilutions for DEDD2 detection in Western blot applications?

For optimal Western blot detection of DEDD2, the following protocol is recommended:

• Use a primary antibody dilution range of 1:500 to 1:2000, with 1:1000 being most commonly effective .

• Secondary antibody (HRP-conjugated anti-rabbit IgG) should be used at 1:10000 dilution .

• Load approximately 25 μg of protein per lane for cell line extracts .

• Use 3% nonfat dry milk in TBST as blocking buffer .

• Develop using an enhanced chemiluminescence (ECL) system with exposure times of approximately 5 seconds for standard detection .

When troubleshooting poor Western blot results, consider adjusting the primary antibody concentration first, followed by extending exposure time or using more sensitive detection systems.

How can I investigate the functional relationship between DEDD2 and intermediate filament degradation during apoptosis?

To investigate the role of DEDD2 in intermediate filament degradation during apoptosis, consider the following methodological approach:

• Track DEDD2 localization dynamics during apoptosis progression:

  • Perform time-course immunofluorescence studies after inducing apoptosis with staurosporine (STS) or death receptor ligands like TRAIL

  • Co-stain for DEDD2 and keratin 8/18 to observe their association at different stages

  • Document the transition from filamentous structures to intracellular inclusions

• Analyze biochemical interactions:

  • Perform co-immunoprecipitation experiments using detergents like Empigen BB to solubilize cytokeratins

  • Investigate whether DEDD2 directly interacts with intermediate filaments or requires other adapter proteins

  • Compare DEDD2 binding to both intact and caspase-cleaved keratins

• Manipulate DEDD2 expression to assess functional impact:

  • Overexpress DEDD2 and measure effects on mitochondrial membrane potential (ΔΨm) using rhodamine 123 staining and PI co-staining

  • Create DEDD2 knockdown cells and analyze keratin cleavage patterns during apoptosis

  • Express dominant-negative DEDD2 constructs and assess their effect on caspase-3 activation and subsequent keratin reorganization

• Utilize ultrastructural analysis:

  • Employ electron microscopy with immunogold labeling to visualize DEDD2 association with intermediate filaments at high resolution

  • Use differently sized gold particles to simultaneously detect DEDD2 and keratins in apoptotic inclusion bodies

How can I assess the potential redundancy or complementation between DEDD and DEDD2 in experimental systems?

Investigating the functional redundancy between DEDD and DEDD2 requires sophisticated experimental approaches:

• Generate single and double knockout/knockdown models:

  • Create DEDD-/-, DEDD2-/-, and DEDD/DEDD2 double knockout cell lines using CRISPR-Cas9

  • Alternatively, use siRNA or shRNA approaches for transient or stable knockdown

• Perform rescue experiments:

  • Re-express DEDD in DEDD-/- cells to assess normalization of phenotypes such as proliferation rates and cell cycle distribution

  • Express DEDD2 in DEDD-/- cells to determine if DEDD2 can complement DEDD functions

  • Create chimeric proteins containing domains from both proteins to identify functional elements

• Compare cellular phenotypes:

  • Analyze proliferation rates and cell cycle distribution in different knockout/knockdown combinations

  • Measure the doubling time of each cell population (e.g., DEDD+/+ cells had doubling times of 45.8 ± 6.5h compared to 37.5 ± 2.5h in DEDD-/- cells)

  • Use BrdU incorporation analyses to determine cell cycle stage distribution

• Examine apoptotic responses:

  • Compare sensitivity to apoptotic stimuli between wild-type, single knockout, and double knockout cells

  • Measure caspase activation kinetics in different genetic backgrounds

  • Analyze intermediate filament degradation patterns in each condition

What approaches can be used to investigate tissue-specific differences in DEDD2 expression and modification?

To investigate tissue-specific differences in DEDD2 expression and modification:

• Analyze expression patterns across tissues:

  • Perform Western blot analysis of different tissue extracts to detect DEDD2 expression levels

  • Compare with DEDD expression, which shows tissue-specific ubiquitination patterns (kidney predominantly expresses unmodified DEDD, while tongue and lung contain mainly mono- and diubiquitinated DEDD, respectively)

  • Use RT-qPCR to quantify DEDD2 mRNA levels across tissues

• Characterize post-translational modifications:

  • Investigate whether DEDD2 undergoes ubiquitination similar to DEDD

  • Analyze changes in solubility of DEDD2 during apoptosis to determine if it transitions from detergent-soluble to detergent-insoluble fractions like DEDD

  • Examine tissue-specific differences in these modifications

• Investigate regulation of expression:

  • Study cell type-specific expression patterns (e.g., higher expression in T cells compared to dendritic cells)

  • Analyze stimulation-dependent changes in expression (e.g., upregulation by LPS or TNF-α in DCs, downregulation by PHA or IL-2 in T cells)

  • Identify transcription factors that regulate DEDD2 expression

• Explore functional consequences of tissue-specific differences:

  • Compare apoptotic susceptibility between cells/tissues with different DEDD2 expression levels

  • Analyze correlation between DEDD2 expression pattern and intermediate filament composition in different tissues

How can I optimize DEDD2 antibody validation for reproducible research applications?

For rigorous DEDD2 antibody validation:

• Establish specificity through multiple approaches:

  • Compare staining patterns with multiple antibodies targeting different DEDD2 epitopes

  • Perform peptide competition assays to confirm epitope specificity

  • Validate antibody performance in DEDD2 knockout or knockdown systems

  • Test cross-reactivity with DEDD and other DED-containing proteins

• Characterize performance across applications:

  • Determine optimal conditions for each application (WB, IF, IP, ELISA)

  • Document lot-to-lot variations by testing multiple antibody batches

  • Establish standardized positive and negative controls for each application

• Document species reactivity:

  • Test antibody performance in human, mouse, and rat samples

  • Note any species-specific differences in recognition patterns or required conditions

  • Verify epitope conservation across species through sequence alignment

• Create a comprehensive validation package:

  • Maintain detailed records of all validation experiments

  • Include positive and negative controls in all experimental procedures

  • Consider publishing validation data to improve reproducibility in the field

What is the best experimental approach to study DEDD2 dynamics during different stages of apoptosis?

To study DEDD2 dynamics during apoptosis progression:

• Use a combination of time-course analyses and imaging techniques:

  • Induce apoptosis with different stimuli (staurosporine, TRAIL, etoposide) to determine pathway-specific effects

  • Perform time-lapse imaging with fluorescently-tagged DEDD2 in live cells

  • Collect samples at defined time points (0h, 1h, 2h, 4h after apoptosis induction) for fixed-cell analyses

• Correlate DEDD2 dynamics with apoptotic markers:

  • Co-stain for active caspase-3, cleaved keratin 18, and DEDD2

  • Monitor nuclear fragmentation, cytoskeletal reorganization, and formation of apoptotic bodies

  • Assess mitochondrial membrane potential changes using Rhodamine 123 staining

• Analyze biochemical changes:

  • Track changes in DEDD2 solubility during apoptosis progression

  • Monitor potential post-translational modifications of DEDD2

  • Investigate protein-protein interaction dynamics using co-immunoprecipitation at different apoptotic stages

• Use high-resolution microscopy techniques:

  • Apply confocal microscopy to track DEDD2 movement from nucleoli to cytoplasmic filaments to inclusion bodies

  • Employ electron microscopy with immunogold labeling for ultrastructural analysis

  • Consider super-resolution techniques to analyze co-localization with cytoskeletal elements in detail

How can I distinguish between nuclear and cytoplasmic functions of DEDD2 in my experiments?

To distinguish between nuclear and cytoplasmic DEDD2 functions:

• Create location-restricted DEDD2 variants:

  • Generate nuclear localization signal (NLS) mutants to restrict DEDD2 to the cytoplasm

  • Create nuclear export signal (NES) mutants or add strong NLS sequences to ensure nuclear retention

  • Verify proper localization using immunofluorescence microscopy

• Perform subcellular fractionation studies:

  • Isolate nuclear, cytoplasmic, and cytoskeletal fractions

  • Analyze DEDD2 distribution across fractions in normal and apoptotic conditions

  • Identify compartment-specific binding partners through mass spectrometry analysis of co-immunoprecipitated proteins

• Investigate function-specific interactions:

  • Test nuclear interactions with transcription factors and potential regulation of GTF3C3

  • Examine cytoplasmic interactions with caspases (CASP8, CASP10) and intermediate filaments

  • Determine if DEDD2 shuttles between compartments in response to specific signals

• Analyze phenotypic consequences of compartment-restricted mutants:

  • Assess effects on apoptosis induction and progression

  • Evaluate impact on intermediate filament organization

  • Measure transcriptional changes associated with nuclear vs. cytoplasmic DEDD2 localization

What experimental controls should be included when studying DEDD2 phosphorylation or ubiquitination status?

When investigating DEDD2 post-translational modifications:

• Include appropriate positive controls:

  • For ubiquitination studies, co-transfect HA-tagged ubiquitin with DEDD2 expression constructs as performed with DEDD

  • Include known ubiquitinated proteins as positive controls

  • Use proteasome inhibitors (e.g., MG132) to accumulate ubiquitinated proteins

• Implement negative controls:

  • Use DEDD2 mutants lacking putative modification sites

  • Include samples treated with deubiquitinating enzymes or phosphatases

  • Compare with related proteins (DEDD) that undergo similar modifications

• Verify specificity of detection:

  • Use antibodies specific for different ubiquitin linkages (K48, K63) or phosphorylated residues

  • Confirm modification sites through mass spectrometry

  • Perform immunoprecipitation under denaturing conditions to eliminate non-covalent interactions

• Include functional assessment:

  • Determine how modifications affect DEDD2 localization

  • Assess impact on protein-protein interactions

  • Evaluate functional consequences on apoptosis induction or cytoskeletal reorganization

What are the common pitfalls in DEDD2 detection and how can they be overcome?

Common challenges in DEDD2 detection and their solutions:

• Low signal-to-noise ratio in immunofluorescence:

  • Optimize fixation and permeabilization conditions (consider epitope masking)

  • Increase antibody concentration or incubation time

  • Note that DEDD2 epitopes may become more accessible during apoptosis, similar to DEDD

  • Use signal amplification methods like tyramide signal amplification

• Poor antibody specificity:

  • Validate antibodies using DEDD2 knockout or knockdown cells

  • Perform peptide competition assays

  • Test multiple antibodies targeting different epitopes

  • Consider using tagged DEDD2 constructs for initial characterization

• Difficulties in solubilizing DEDD2:

  • DEDD2 may become detergent-insoluble during apoptosis, similar to DEDD

  • Use stronger extraction buffers containing Empigen BB, which can solubilize approximately 40% of cellular cytokeratins

  • Consider separate analysis of detergent-soluble and detergent-insoluble fractions

• Challenges in detecting post-translational modifications:

  • Use phosphatase or deubiquitinase inhibitors in lysis buffers

  • Consider specialized enrichment techniques for modified proteins

  • Apply denaturing conditions during immunoprecipitation to maintain modifications

How can I optimize conditions for co-immunoprecipitation studies involving DEDD2?

For successful co-immunoprecipitation of DEDD2 and its interaction partners:

• Optimize lysis conditions:

  • Choose detergents carefully - milder detergents (0.5% NP-40, 0.5% Triton X-100) for preserving protein-protein interactions

  • For interactions with cytoskeletal components, consider specialized detergents like Empigen BB that can solubilize intermediate filaments

  • Include protease and phosphatase inhibitors to preserve protein integrity

• Select appropriate antibodies:

  • Use antibodies validated for immunoprecipitation applications

  • Consider epitope accessibility in protein complexes

  • For difficult targets, consider using tagged DEDD2 constructs and anti-tag antibodies

• Control for specificity:

  • Include isotype-matched control antibodies

  • Use cells lacking DEDD2 expression as negative controls

  • Perform reciprocal co-IPs when possible to confirm interactions

• Adjust binding and washing conditions:

  • Optimize antibody-to-lysate ratios

  • Adjust binding time and temperature (4°C overnight often works well)

  • Determine appropriate washing stringency to remove non-specific interactions while preserving specific ones

What are the recommended approaches for quantifying DEDD2 expression levels in different experimental conditions?

For accurate quantification of DEDD2 expression:

• Protein level quantification:

  • Western blot analysis with proper loading controls (β-actin, GAPDH)

  • Use infrared fluorescence-based detection systems for wider linear range

  • Consider ELISA development for higher throughput quantification

  • Include a standard curve with recombinant DEDD2 for absolute quantification

• mRNA level quantification:

  • Use RT-qPCR with validated primers and appropriate reference genes

  • Normalize to multiple reference genes for more reliable results

  • Consider digital PCR for absolute quantification without standard curves

  • RNA-seq for genome-wide expression analysis in context with other genes

• Single-cell analysis:

  • Flow cytometry for population-level analysis of DEDD2 expression

  • Immunofluorescence microscopy with image analysis for subcellular localization

  • Single-cell RNA-seq for transcriptional heterogeneity

• Data analysis considerations:

  • Use appropriate statistical tests based on data distribution

  • Perform replicate experiments (biological and technical)

  • For complex experimental designs, consider analysis of variance (ANOVA) or mixed-effects models

  • Present normalized data alongside raw values when possible

How can I apply CRISPR-Cas9 technology to study DEDD2 function in apoptotic pathways?

Implementing CRISPR-Cas9 approaches for DEDD2 functional studies:

• Generate knockout models:

  • Design guide RNAs targeting early exons of DEDD2

  • Create cell lines with complete DEDD2 knockout

  • Generate DEDD/DEDD2 double knockout lines to assess functional redundancy

  • Verify knockouts by sequencing, Western blot, and functional assays

• Create knock-in models for detailed mechanistic studies:

  • Generate fluorescently tagged DEDD2 at endogenous loci for live-cell imaging

  • Create specific point mutations to disrupt functional domains (DED, NLS)

  • Introduce modifications that prevent ubiquitination or other post-translational modifications

  • Develop conditional knockout models using floxed alleles

• Perform genetic screens:

  • Use CRISPR libraries to identify genes that interact with DEDD2 in apoptotic pathways

  • Screen for suppressors or enhancers of DEDD2-dependent phenotypes

  • Identify synthetic lethal interactions with DEDD2 deletion

• Comparative analysis with DEDD:

  • Create isogenic cell lines with DEDD knockout, DEDD2 knockout, and double knockout

  • Assess proliferation rates, cell cycle distribution, and apoptotic responses

  • Measure the impact on intermediate filament degradation during apoptosis

What high-throughput approaches can be used to identify novel DEDD2 interaction partners?

Advanced methods for identifying DEDD2 interaction networks:

• Proximity-based labeling approaches:

  • BioID or TurboID fusion proteins to identify proteins in close proximity to DEDD2

  • APEX2 tagging for spatial-temporal mapping of DEDD2 interactome

  • Compare interactomes in normal versus apoptotic conditions

• Mass spectrometry-based interactomics:

  • Immunoprecipitation coupled with mass spectrometry (IP-MS)

  • Cross-linking mass spectrometry (XL-MS) to capture transient interactions

  • Quantitative approaches like SILAC or TMT labeling to compare interaction changes during apoptosis progression

• Protein microarray screening:

  • Probe protein arrays with recombinant DEDD2 to identify direct binding partners

  • Use domain-specific constructs to map interaction interfaces

  • Compare DEDD and DEDD2 binding profiles to identify shared and unique partners

• Functional genomics approaches:

  • CRISPR screens in DEDD2-dependent phenotypic assays

  • Synthetic genetic array analysis to identify genetic interactions

  • Transcriptomic profiling of DEDD2 knockout versus wildtype cells to identify downstream effectors