DNM2 Antibody, HRP conjugated

What are the recommended applications and dilutions for DNM2 antibody, HRP conjugated?

DNM2 antibody, HRP conjugated is primarily optimized for ELISA applications. Based on manufacturer specifications, HRP-conjugated variants (such as CSB-PA007079LB01HU and CSB-PA007079LB11HU) require application-specific dilutions:

For comparison, unconjugated DNM2 antibodies are used across multiple applications with the following dilution ranges:

Always establish optimal concentrations through titration experiments in your specific experimental system, as sample type and detection methods significantly influence optimal antibody concentration .

How do you validate the specificity of a DNM2 antibody?

A multi-faceted approach to validating DNM2 antibody specificity should include:

• Western blot verification: Confirm detection of a single band at the expected molecular weight (98-100 kDa) across multiple DNM2-expressing samples (e.g., HeLa cells, HEK-293 cells, brain tissue)

• Positive and negative controls:

  • Positive controls: Brain tissue samples (human, mouse, rat) which express high levels of DNM2

  • Negative controls: DNM2 knockdown/knockout samples or irrelevant cell lines

• Cross-reactivity testing: For antibodies claiming multi-species reactivity, validate using samples from each claimed species (human, mouse, rat, pig, rabbit)

• Application-specific validation: For HRP-conjugated antibodies specifically used in ELISA, perform blocking experiments with recombinant DNM2 protein

• For therapeutic research: Verify antibody recognition of mutant DNM2 forms relevant to disease models (R465W mutation in AD-CNM, K562E mutation in CMT)

What is the specificity of DNM2 antibodies across species?

DNM2 antibodies show varying cross-reactivity profiles that must be verified for each research application:

What is the difference between ubiquitous DNM2 and muscle-specific DNM2 isoforms?

DNM2 exists in two principal isoforms with distinct expression patterns and potential functional differences:

The tissue-specific expression pattern of M-DNM2 may contribute to the tissue-specific pathology observed in DNM2-related diseases despite the ubiquitous expression of DNM2. This distinction is particularly relevant for therapeutic approaches targeting specific DNM2 isoforms .

How can DNM2 antibodies be used to distinguish between different isoforms of DNM2?

Distinguishing between DNM2 isoforms requires specialized methodological approaches:

• Isoform-specific antibodies: Develop antibodies against the exon 12b peptide sequence unique to M-DNM2, as described in research where "a rabbit antibody was raised against the exon 12b peptide and detects specifically mouse and human M-DNM2"

• Combinatorial approaches when isoform-specific antibodies are unavailable:

  • RT-PCR with primers specific for each isoform followed by protein analysis

  • 2D gel electrophoresis to separate isoforms based on slight differences in molecular weight and isoelectric point

  • Immunoprecipitation with pan-DNM2 antibodies followed by mass spectrometry to identify isoform-specific peptides

• Tissue-specific expression analysis: Compare tissues with known differential expression (e.g., skeletal muscle expressing both isoforms versus non-muscle tissues expressing only Ub-DNM2)

• Validation methodology:

  • Use knockout/knockdown models specific to each isoform

  • Apply allele-specific silencing approaches that target one isoform but not the other

What methodological considerations are important when using DNM2 antibodies in studies of neuromuscular diseases?

When investigating DNM2-related neuromuscular diseases, several critical methodological factors must be addressed:

• Mutation-specific considerations:

  • CNM mutations primarily affect the auto-inhibitory interface

  • CMT mutations concentrate in the lipid binding loops of the PH domain

  • These structural differences may affect antibody binding and protein detection efficiency

• Age-dependent expression patterns:

  • DNM2 protein shows physiological decrease with age in wild-type mice

  • Pathological accumulation of DNM2 protein occurs with age in disease models

  • Time course studies should include age-matched controls and measure DNM2 levels at multiple timepoints

• Therapeutic monitoring protocols:

  • For allele-specific silencing therapies: use RT-PCR with EcoNI restriction enzyme digestion to discriminate between wild-type and mutated alleles

  • For protein expression studies: apply densitometric analysis of Western blots with appropriate loading controls

  • For long-term studies: monitor DNM2 expression patterns for at least 1 year after therapeutic intervention

• Tissue-specific sampling strategy:

  • Include both muscle and nerve tissue samples in CMT studies

  • Focus on skeletal muscle in CNM studies

  • Consider cardiac muscle samples when studying the muscle-specific isoform M-DNM2

What considerations are important when using DNM2 antibodies for investigating the interaction between DNM2 and BIN1?

The DNM2-BIN1 interaction represents a critical regulatory mechanism with therapeutic implications for neuromuscular diseases. When investigating this interaction:

• Domain-specific antibody selection:

  • BIN1's SH3 domain interacts with DNM2's PRD domain

  • Avoid antibodies targeting these interaction domains if studying the native interaction

  • For disruption studies, specifically target these domains

• Consider phosphorylation status:

  • Phosphorylation of DNM2-S848 in the PRD decreases binding affinity to BIN1-SH3

  • Include phosphatase inhibitors in lysis buffers when preserving native interaction state

  • Use phosphomimetic mutants (e.g., Dyn2 S848E) as controls for disrupted interaction

• Disease-specific interaction patterns:

  • BIN1 reduction ameliorates DNM2-related Charcot-Marie-Tooth neuropathy

  • Increasing BIN1 exacerbates phenotypes in DNM2 K562E/+ CMT mouse model

  • The opposite therapeutic approach (increasing BIN1) rescues centronuclear myopathy in other DNM2 mutation models

• Experimental approaches:

  • GST pulldown assays with GST-tagged PRD of DNM2 and His-Bin1 proteins

  • In vitro fission assays to assess functional consequences of the interaction

  • Liposome tubulation assays to visualize Bin1-DNM2 assembly structures

How can DNM2 antibodies be used to investigate the role of DNM2 in endocytosis?

Investigating DNM2's critical role in endocytosis requires multiple methodological approaches:

• Colocalization studies:

  • Immunofluorescence with DNM2 antibodies alongside markers for:

    • Clathrin-mediated endocytosis components

    • Clathrin-independent endocytic pathways

    • Vesicular trafficking markers (early endosomes, recycling endosomes)

  • Use high-resolution microscopy techniques (STED, STORM) for precise localization

• Functional endocytosis assays:

  • Transferrin uptake assays following DNM2 knockdown/mutation

  • PDGF-stimulated macropinocytosis assessment

  • Combine with DNM2 antibody staining at fixed timepoints

• Biochemical approaches:

  • Membrane fractionation followed by Western blotting with DNM2 antibodies

  • GTPase activity assays using purified DNM2

  • Liposome-binding assays to assess membrane association properties

• Advanced interaction studies:

  • Proximity ligation assays between DNM2 and endocytic proteins

  • FRET/FLIM studies with labeled DNM2 antibodies

  • Immunoprecipitation of DNM2 during various stages of endocytosis

How do you optimize DNM2 antibody use for detecting post-translational modifications like phosphorylation?

Detecting DNM2 post-translational modifications requires specific optimization strategies:

• Phosphorylation-site specific approaches:

  • Use phospho-specific antibodies targeting known sites (e.g., Ser848 in PRD domain)

  • Implement phosphatase inhibitors in lysis buffers (sodium orthovanadate, sodium fluoride)

  • Include lambda phosphatase treatment controls to confirm phospho-specific detection

• Validation using mutational analysis:

  • Compare wild-type DNM2 with phosphomimetic (Ser→Glu) mutants

  • Assess functional consequences using GTPase activity assays

  • Use phospho-deficient (Ser→Ala) mutants as negative controls

• Functional correlation studies:

  • Link phosphorylation status to interaction with binding partners (e.g., BIN1)

  • Analyze impact on membrane fission activities

  • Connect phosphorylation patterns to subcellular localization

• Technical considerations for Western blotting:

  • Use Phos-tag™ acrylamide gels for enhanced separation of phosphorylated species

  • Employ ELISA-based methods with HRP-conjugated secondary antibodies for quantification

  • Consider multiplexed approaches to simultaneously detect total and phosphorylated DNM2

How can DNM2 antibodies be used in therapeutic research for DNM2-related diseases?

DNM2 antibodies serve crucial functions in therapeutic research for DNM2-related diseases:

• Monitoring therapeutic efficacy:

  • Quantify DNM2 levels following RNA interference therapies

  • Assess allele-specific silencing efficiency through RT-PCR and Western blot

  • Track long-term protein expression patterns after single interventions

• Disease mechanism investigation:

  • Determine age-related DNM2 accumulation in disease models

  • Assess isoform-specific contributions to pathology

  • Evaluate tissue-specific responses to therapy

• Methodological approaches for therapeutic assessment:

  • RT-PCR with EcoNI digestion to discriminate between wild-type and mutated alleles

  • Densitometric analysis of Western blots to quantify protein levels

  • Immunohistochemical analysis to assess DNM2 distribution in target tissues

• Therapeutic strategy validation:

  • For DNM2-CMT: BIN1 reduction therapeutic approach

  • For DNM2-CNM: Mutant-specific RNA interference

  • Different therapeutic strategies required despite mutations in the same gene

• AAV-based therapy monitoring:

  • Track viral genome distribution in target tissues

  • Quantify shRNA expression levels

  • Monitor long-term therapeutic effects (up to 1 year after single injection)

What are the challenges in using DNM2 antibodies to study the role of DNM2 in GTPase activity?

Investigating DNM2's GTPase activity presents several methodological challenges:

• Technical limitations:

  • Standard GTPase assays (malachite green assay) require purified proteins

  • Immunoprecipitated DNM2 may retain binding partners that affect activity

  • Antibody binding may directly interfere with GTPase domain function

• Methodological solutions:

  • Use multiple antibodies targeting different epitopes

  • Complement with tagged recombinant DNM2 constructs

  • Develop assays for immunoprecipitated complexes

• Functional assay considerations:

  • Liposome-stimulated GTPase activity measurement

  • Assessment of how binding partners (e.g., BIN1) regulate activity

  • Correlation between membrane binding and GTPase activity

• In vitro vs. cellular approaches:

  • In vitro: Purified DNM2 with liposomes containing 5% PI(4,5)P₂

  • Cellular: Immunofluorescence combined with functional endocytosis assays

  • Correlative approaches linking localization to activity

What considerations are important for using DNM2 antibodies in neutropenia research related to Charcot-Marie-Tooth disease?

Research on DNM2-related neutropenia in Charcot-Marie-Tooth disease requires specific methodological considerations:

• Age and sex considerations:

  • Female mice with het Dnm2 haploinsufficiency develop neutropenia as they age

  • This mimics human clinical presentation of chronic idiopathic neutropenia predominant in middle-aged women

  • Age-matched controls are essential for accurate interpretation

• Tissue-specific analyses:

  • Bone marrow neutrophil retention assessment

  • Investigation of CXCR4 surface expression on neutrophils

  • Splenic germinal center evaluation

• Combined methodological approaches:

  • Flow cytometry for neutrophil quantification

  • Immunohistochemistry for tissue distribution analysis

  • Western blotting for protein expression quantification

• Therapeutic research considerations:

  • DNM2 inhibition affects neutrophil migration

  • Age-specific intervention timing may be critical

  • Different therapeutic approaches may be needed for neurological vs. hematological manifestations