DNM1L (Ab-637) Antibody

What is DNM1L and what are its primary cellular functions?

DNM1L (Dynamin-1-like protein), also known as DRP1 (Dynamin-related protein 1), is a GTPase that plays a crucial role in mitochondrial and peroxisomal division. It mediates membrane fission through oligomerization into membrane-associated tubular structures that wrap around scission sites to constrict and sever mitochondrial membranes through a GTP hydrolysis-dependent mechanism . The protein functions in:

• Regulating mitochondrial dynamics (fission/fusion balance)

• Facilitating developmentally regulated apoptosis

• Supporting normal brain development, particularly in the cerebellum

• Suppressing oxidative damage in postmitotic neurons

• Controlling mitochondrial fission during mitosis

DNM1L's recruitment to mitochondrial membranes is mediated by specific membrane receptors such as MFF, MIEF1, and MIEF2 in a GTP-dependent manner .

What are the key specifications of the DNM1L (Ab-637) Antibody?

The DNM1L (Ab-637) Antibody is a rabbit polyclonal antibody with the following specifications:

Note: At least four isoforms of DNM1L are known to exist; this antibody typically detects the two longest isoforms .

What are the main research applications for DNM1L (Ab-637) Antibody?

DNM1L (Ab-637) Antibody has been utilized in multiple research applications, particularly focusing on mitochondrial dynamics and associated pathologies:

• Neurodegenerative research: Investigating mitochondrial dynamics in neuronal cells, particularly in models of glaucoma and other neurodegenerative conditions

• Cancer research: Studying altered mitochondrial dynamics in cancer, particularly in lung adenocarcinoma where DNM1L is overexpressed

• Cardiovascular research: Examining mitochondrial fission in cardiomyopathy models and other cardiac conditions

• Cell biology: Investigating fundamental aspects of mitochondrial dynamics, fission/fusion balance, and quality control mechanisms

• Immunological research: Exploring the role of DNM1L in regulating immune cell infiltration in tumor microenvironments

The antibody has been validated for Western blotting (WB) and ELISA applications across all sources, with some formulations also suitable for immunofluorescence (IF) and immunocytochemistry (ICC) .

What are the optimal conditions for using DNM1L (Ab-637) Antibody in Western blotting?

For optimal Western blotting using DNM1L (Ab-637) Antibody, consider the following methodological recommendations:

• Sample preparation:

  • Use RIPA buffer with protease and phosphatase inhibitors for protein extraction

  • For mitochondrial enrichment, consider subcellular fractionation techniques

• Gel electrophoresis:

  • Use 8-10% SDS-PAGE gels (DNM1L has a predicted MW of 81 kDa, observed at approximately 80 kDa)

  • Load 20-40 μg of total protein per lane

• Transfer and blocking:

  • Transfer to PVDF membrane (preferred over nitrocellulose for this protein)

  • Block with 5% non-fat dry milk in TBST for 1 hour at room temperature

• Antibody incubation:

  • Primary antibody dilution: 1:500-1:3000 in blocking buffer

  • Incubate overnight at 4°C with gentle rocking

  • Secondary antibody: Anti-rabbit HRP at 1:5000-1:10000 dilution

• Detection:

  • Enhanced chemiluminescence (ECL) is sufficient for most applications

  • Expected band: ~80 kDa (main band); may detect additional bands representing different phosphorylation states or splice variants

Troubleshooting tip: If background is high, consider using BSA instead of milk for blocking and antibody dilution, or increase washing steps with TBST.

How can I optimize DNM1L (Ab-637) Antibody for immunofluorescence studies?

For optimal immunofluorescence staining with DNM1L (Ab-637) Antibody:

• Fixation and permeabilization:

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

  • Permeabilize with 0.1% Triton X-100 for 5-10 minutes (critical for accessing mitochondrial proteins)

• Blocking:

  • Block with 5% normal goat serum in PBS for 1 hour at room temperature

• Antibody incubation:

  • Primary antibody: Start at 20 μg/mL and titrate if needed

  • Incubate overnight at 4°C in a humidified chamber

  • Secondary antibody: Anti-rabbit conjugated with fluorophore of choice at 1:200-1:500

• Counterstaining:

  • MitoTracker or TOMM20 antibody for mitochondrial colocalization

  • DAPI for nuclear staining

• Mounting and imaging:

  • Mount with anti-fade mounting medium

  • Use confocal microscopy for optimal resolution of mitochondrial structures

For visualizing mitochondrial dynamics, consider live-cell imaging with fluorescently tagged DNM1L constructs as a complementary approach to fixed-cell immunofluorescence .

What controls should be included when using DNM1L (Ab-637) Antibody in experimental designs?

Robust experimental designs using DNM1L (Ab-637) Antibody should include the following controls:

• Positive controls:

  • Cell lines with known DNM1L expression (e.g., HeLa cells, HEK293 cells)

  • Tissues with high mitochondrial content (e.g., heart, brain)

  • Recombinant DNM1L protein (for antibody validation)

• Negative controls:

  • DNM1L knockout or knockdown cells/tissues (siRNA or CRISPR-generated)

  • Isotype control (rabbit IgG at same concentration)

  • Secondary antibody only control

• Specificity controls:

  • Preabsorption of antibody with immunizing peptide

  • Use of multiple antibodies targeting different epitopes of DNM1L

  • Comparison with commercially available validated anti-DNM1L antibodies

• Experimental manipulation controls:

  • Treatment with mitochondrial fission inducers (e.g., FCCP, rotenone)

  • Mitochondrial fusion promoters (e.g., Mdivi-1 as DNM1L inhibitor)

  • Phosphorylation state controls (e.g., AMPK activators for Ser637 phosphorylation)

Including these controls helps validate antibody specificity and ensures accurate interpretation of experimental results, particularly in complex systems where mitochondrial dynamics are being studied.

How has DNM1L (Ab-637) Antibody been used in glaucoma research?

DNM1L (Ab-637) Antibody has been instrumental in glaucoma research, particularly in identifying DNM1L as a therapeutic target. Key methodological approaches and findings include:

• Antibody-based therapeutic approach:

  • Intravitreal application of anti-DNM1L antibody demonstrated protective effects for retinal ganglion cells (RGCs) and their axons in the retinal nerve fiber layer (RNFL)

  • This treatment improved retinal functionality as measured by electroretinography (Ganzfeld ERG)

• Mechanism investigation:

  • Western blot analysis using DNM1L antibodies revealed altered phosphorylation of DNM1L and changed expression of apoptosis-related proteins following treatment

  • Analysis showed significantly increased XIAP expression (fold-change: 1.10 ± 0.12) in anti-DNM1L antibody-treated samples compared to IgG controls (fold-change: 0.69 ± 0.15, p < 0.05)

• Proteomics integration:

  • Mass spectrometry analysis identified 28 up-regulated and 21 down-regulated proteins after anti-DNM1L antibody treatment

  • Protein pathway analysis revealed three main interacting clusters: vesicle traffic-associated (NSF, SNCA, ARF1), mitochondrion-associated (HSP9A, SLC25A5/ANT2, GLUD1), and cytoskeleton-associated (MAP1A) signaling pathways

• Quantitative measurements:

  • Optical Coherence Tomography (OCT) showed that anti-DNM1L antibody treatment resulted in approximately 15% higher RNFLT in temporal superior (TS) and temporal inferior (TI) regions compared to controls

These findings highlight that targeting DNM1L with antibodies represents a promising therapeutic approach for glaucoma, suggesting potential applications for testing DNM1L antibodies in other neurodegenerative conditions involving mitochondrial dysfunction.

What role does DNM1L play in cancer research, and how is the antibody utilized?

DNM1L has emerged as an important factor in cancer research, particularly in lung adenocarcinoma (LUAD). The DNM1L (Ab-637) Antibody has been utilized in several methodological approaches:

• Expression analysis in tumor tissues:

  • Immunohistochemistry and Western blot analyses have demonstrated that DNM1L is overexpressed in LUAD compared to healthy control tissues

  • The antibody has been used to quantify expression levels across different tumor grades, stages, and in correlation with clinical variables

• Functional studies in cancer cells:

  • DNM1L antibodies have been used to investigate its role in regulating proliferation and migration of LUAD cells

  • Knockdown and inhibition studies followed by antibody-based detection have revealed DNM1L's mechanistic contributions to cancer progression

• Immune microenvironment analysis:

  • Research using DNM1L antibodies has uncovered its potential role in regulating immune cell infiltration in the tumor microenvironment

  • Studies have shown correlation between DNM1L expression (detected via antibodies) and the abundance of specific immune cell populations

• Prognostic biomarker validation:

  • DNM1L antibody-based detection methods have been used to establish its value as a prognostic biomarker

  • Expression levels correlated with survival outcomes in Kaplan-Meier analyses

This research suggests that DNM1L plays a multifaceted role in cancer biology, potentially serving as both a prognostic biomarker and therapeutic target. The antibody has been instrumental in elucidating these functions, demonstrating that beyond mitochondrial dynamics, DNM1L may influence cancer progression through effects on cell proliferation and the immune microenvironment.

What insights has DNM1L (Ab-637) Antibody provided in cardiovascular disease research?

DNM1L (Ab-637) Antibody has contributed significantly to cardiovascular research, revealing important connections between mitochondrial dynamics and heart disease:

These findings highlight DNM1L's critical role in cardiovascular pathophysiology and suggest that targeting mitochondrial dynamics could represent a novel therapeutic approach for heart diseases. The antibody has been essential for tracking both expression levels and functional changes in DNM1L across various cardiac conditions.

How can I troubleshoot non-specific binding when using DNM1L (Ab-637) Antibody in Western blotting?

Non-specific binding is a common challenge when using DNM1L (Ab-637) Antibody. Here's a methodological approach to troubleshooting:

• Optimize antibody dilution:

  • Titrate the antibody beyond the recommended 1:500-1:3000 range

  • Prepare a dilution series (e.g., 1:500, 1:1000, 1:2000, 1:3000) to identify optimal signal-to-noise ratio

• Adjust blocking conditions:

  • Try different blocking agents: 5% non-fat milk, 5% BSA, or commercial blocking buffers

  • Increase blocking time from 1 hour to 2-3 hours at room temperature

  • Add 0.1-0.3% Tween-20 to blocking buffer to reduce hydrophobic interactions

• Optimize washing steps:

  • Increase number of washes (5-6 times for 5-10 minutes each)

  • Use higher concentration of Tween-20 in wash buffer (0.1-0.2%)

  • Consider using TBS instead of PBS if phosphorylated DNM1L is the target

• Sample preparation refinements:

  • Include additional protease inhibitors in lysis buffer

  • Try different detergents in lysis buffer (e.g., CHAPS instead of Triton X-100)

  • Consider using mitochondrial fraction enrichment to increase target protein concentration

• Validate with additional controls:

  • Use DNM1L knockdown samples as negative controls

  • Include both positive (cells with high DNM1L expression) and negative controls

  • Pre-absorb antibody with immunizing peptide to confirm specificity of bands

If persistent non-specific bands appear at ~60-65 kDa, these may represent degradation products of DNM1L or alternative splice variants. Multiple bands around 80-85 kDa often represent different phosphorylation states of DNM1L, particularly at Ser616 and Ser637, which regulate its activity .

How should I interpret changes in DNM1L phosphorylation patterns in my experiments?

Interpreting DNM1L phosphorylation patterns requires careful consideration of its regulatory mechanisms:

• Key phosphorylation sites and their significance:

  • Ser616 phosphorylation (human; Ser635 in mouse):

    • Promotes mitochondrial fission activity

    • Increased in mitosis and by various stressors

    • Mediated by CDK1/cyclin B, ERK1/2, PKCδ, and CaMKIα

  • Ser637 phosphorylation (human; Ser656 in mouse):

    • Inhibits DNM1L activity and mitochondrial translocation

    • Mediated by PKA

    • Dephosphorylated by calcineurin during apoptosis or calcium flux

• Experimental interpretation guidelines:

  • Always normalize phospho-DNM1L to total DNM1L levels

  • Compare pSer616/pSer637 ratio as an indicator of fission/fusion balance

  • Consider subcellular localization - cytosolic vs. mitochondria-associated DNM1L

• Common patterns and their meaning:

  Pattern	Biological Interpretation	Example Contexts
  ↑ pSer616, ↔ pSer637	Enhanced fission activity	Mitosis, cellular stress, hypoxia
  ↔ pSer616, ↓ pSer637	Enhanced fission via reduced inhibition	Apoptosis, calcium flux
  ↓ pSer616, ↑ pSer637	Reduced fission, fusion predominance	Starvation, AMPK activation
  ↑ pSer616, ↑ pSer637	Complex regulation, context-dependent	Some pathological states

• Research context examples:

  • In glaucoma models, anti-DNM1L antibody treatment altered DNM1L phosphorylation patterns, correlating with neuroprotection

  • In FCCP-induced stress, wild-type cells showed DNM1L-Ser616 and MAPK/ERK phosphorylation while mutant cells did not, despite similar total protein levels

  • In mitochondrial clearance experiments, knockdown of DNM1L affected mitochondrial network integrity more prominently in mutant MEFs, indicating preexisting impaired DNM1L activation

When interpreting your results, consider that changes in phosphorylation may occur without changes in total DNM1L levels, and alterations in the phosphorylation ratio often provide more insight than absolute changes in individual sites.

How can I differentiate between DNM1L isoforms in my experimental results?

Differentiating between DNM1L isoforms requires careful methodological approaches:

• Understanding DNM1L isoform diversity:

  • At least four isoforms of DNM1L exist due to alternative splicing

  • The DNM1L (Ab-637) Antibody typically detects the two longest isoforms

  • Isoforms differ primarily in the presence/absence of specific domains or insertions

• Western blot strategies for isoform differentiation:

  • Use higher resolution gels (8-10% acrylamide with longer run times)

  • Consider using gradient gels (4-12%) for better separation

  • Run samples longer to maximize band separation

  • Include positive controls for different isoforms when available

• Advanced techniques for isoform identification:

  • 2D gel electrophoresis:

    • First dimension: Isoelectric focusing (IEF)

    • Second dimension: SDS-PAGE

    • Followed by immunoblotting with DNM1L (Ab-637) Antibody

  • RT-PCR analysis in parallel with protein detection:

    • Design primers specific to different splice variants

    • Correlate mRNA expression with protein band patterns

  • Mass spectrometry:

    • Immunoprecipitate with DNM1L (Ab-637) Antibody

    • Perform tryptic digestion and LC-MS/MS

    • Identify peptides unique to specific isoforms

• Interpretation guidelines:

  • The main DNM1L isoform appears at ~80 kDa

  • Shorter isoforms typically appear at 70-78 kDa

  • Post-translational modifications (especially phosphorylation) can cause slight shifts in apparent molecular weight

  • Tissue-specific expression patterns may influence isoform distribution

For most accurate isoform differentiation, consider combining the DNM1L (Ab-637) Antibody with isoform-specific antibodies when available, or use genetic approaches (isoform-specific overexpression or knockdown) as complementary methods to validate band identity.

How can I design experiments to investigate DNM1L's role in mitochondrial dynamics using the Ab-637 antibody?

Designing comprehensive experiments to investigate DNM1L's role in mitochondrial dynamics requires multi-faceted approaches:

• Mitochondrial morphology assessment:

  • Immunofluorescence co-localization:

    • Use DNM1L (Ab-637) Antibody (20 μg/mL) alongside mitochondrial markers (MitoTracker or anti-TOMM20)

    • Quantify colocalization using Pearson's or Mander's coefficient

  • Live-cell imaging:

    • Combine with fluorescently tagged constructs (mito-GFP)

    • Track fission events temporally in relation to DNM1L recruitment

  • Electron microscopy:

    • Use immunogold labeling with DNM1L (Ab-637) Antibody

    • Examine DNM1L localization at constriction sites

• Functional manipulation studies:

  • Pharmacological approaches:

    • Apply mitochondrial fission inducers (FCCP, rotenone) or inhibitors (Mdivi-1)

    • Monitor DNM1L phosphorylation state changes via Western blot

    • Track translocation using fractionation followed by immunoblotting

  • Genetic approaches:

    • Knockdown/knockout DNM1L using siRNA or CRISPR-Cas9

    • Rescue with wild-type or mutant DNM1L variants

    • Assess mitochondrial network changes quantitatively

• Interaction network analysis:

  • Co-immunoprecipitation:

    • Use DNM1L (Ab-637) Antibody to pull down DNM1L and interacting partners

    • Identify novel interactions through mass spectrometry

  • Proximity labeling:

    • Combine with BioID or APEX approaches

    • Validate interactions by immunoblotting with DNM1L (Ab-637) Antibody

• Quantitative assessment metrics:

  • Morphological parameters:

    • Form factor (perimeter²/4π×area)

    • Aspect ratio (major axis/minor axis)

    • Average mitochondrial length and footprint

  • Functional readouts:

    • Mitochondrial membrane potential (TMRM, JC-1)

    • ROS production (MitoSOX)

    • ATP production rate

    • Oxygen consumption rate (Seahorse)

This experimental design framework allows for comprehensive investigation of both static and dynamic aspects of DNM1L function in mitochondrial dynamics, with the DNM1L (Ab-637) Antibody serving as a key tool for detection, localization, and interaction studies.

What are the most effective methods for studying DNM1L phosphorylation state changes in disease models?

Studying DNM1L phosphorylation state changes in disease models requires sophisticated methodological approaches that can detect subtle modifications with high specificity:

• Phospho-specific antibody strategies:

  • Use phospho-specific antibodies for key regulatory sites (pSer616, pSer637) in conjunction with DNM1L (Ab-637) Antibody

  • Calculate phosphorylation ratios (phospho/total DNM1L) to normalize for expression differences

  • Compare pSer616/pSer637 ratio as an indicator of activation state

• Kinase/phosphatase manipulation approaches:

  • Pharmacological interventions:

    • PKA activators (forskolin, cAMP analogs) → increase Ser637 phosphorylation

    • Calcineurin inhibitors (cyclosporin A, FK506) → prevent Ser637 dephosphorylation

    • ERK pathway modulators → affect Ser616 phosphorylation

  • Genetic approaches:

    • Express phosphomimetic (S→D/E) or phosphodeficient (S→A) DNM1L mutants

    • Knockdown specific kinases/phosphatases and assess DNM1L phosphorylation

    • CRISPR-mediated genome editing of phosphorylation sites

• Advanced analytical techniques:

  • Phos-tag SDS-PAGE:

    • Enhanced separation of phosphorylated from non-phosphorylated forms

    • Follow with Western blotting using DNM1L (Ab-637) Antibody

    • Quantify multiple phosphorylation states simultaneously

  • Mass spectrometry-based phosphoproteomics:

    • Immunoprecipitate DNM1L followed by LC-MS/MS

    • Quantify site-specific phosphorylation stoichiometry

    • Identify novel phosphorylation sites

  • Proximity ligation assay (PLA):

    • Detect interactions between DNM1L and kinases/phosphatases in situ

    • Correlate with mitochondrial morphology changes

• Disease model-specific considerations:

  • In glaucoma models, anti-DNM1L antibody treatment significantly altered phosphorylation states, particularly affecting BAD phosphorylation (p112)

  • In LRRK2 mutant models, FCCP-induced stress revealed differential DNM1L-Ser616 and MAPK/ERK phosphorylation patterns between wild-type and mutant cells

  • In heart failure models like the Python mouse, altered DNM1L function affected mitochondrial morphology without obvious aggregation, suggesting subtle regulatory changes

When applying these methods to disease models, time-course experiments are particularly valuable, as phosphorylation changes often precede phenotypic alterations and may represent early therapeutic intervention points.

How can I integrate DNM1L research with broader studies of cellular metabolism and disease pathogenesis?

Integrating DNM1L research into broader investigations of cellular metabolism and disease pathogenesis requires methodological approaches that connect mitochondrial dynamics to downstream cellular processes:

• Metabolic pathway analysis integration:

  • Metabolic flux analysis:

    • Combine DNM1L manipulation with stable isotope tracing (¹³C-glucose, ¹³C-glutamine)

    • Correlate DNM1L activity (detected via Ab-637) with TCA cycle activity and glucose oxidation

    • Measure glycolytic shifts using extracellular acidification rate (ECAR) and oxygen consumption rate (OCR)

  • Mitochondrial function assessment:

    • ATP production capacity in response to DNM1L manipulation

    • Mitochondrial membrane potential changes using potentiometric dyes

    • ROS production using fluorescent indicators correlated with DNM1L activity or phosphorylation state

• Multi-omics integration approaches:

  • Transcriptomics correlation:

    • RNA-seq following DNM1L manipulation or in disease models

    • Pathway analysis to identify gene expression networks affected by altered mitochondrial dynamics

  • Proteomics integration:

    • Compare proteome changes in response to DNM1L manipulation

    • In glaucoma models, anti-DNM1L antibody treatment revealed 28 up-regulated and 21 down-regulated proteins

    • Protein interaction networks showed three main interacting clusters: vesicle traffic-associated, mitochondrion-associated, and cytoskeleton-associated pathways

  • Metabolomics correlation:

    • Identify metabolite changes associated with altered DNM1L function

    • Focus on mitochondrial metabolites (TCA cycle intermediates, acylcarnitines)

• Disease-specific mechanistic studies:

  • Cancer metabolism:

    • DNM1L overexpression in lung adenocarcinoma correlates with altered cellular energetics

    • Investigate connections between mitochondrial fragmentation and the Warburg effect

    • Explore metabolic vulnerabilities created by DNM1L-dependent changes

  • Neurodegeneration mechanisms:

    • In glaucoma, anti-DNM1L antibody treatment protected retinal ganglion cells and their axons

    • Explore how mitochondrial dynamics affects axonal transport and synaptic function

    • Investigate connections to oxidative stress and neuronal bioenergetics

  • Cardiovascular metabolism:

    • In the Python mouse model, DNM1L mutation led to approximately 50% reduction in ATP and total adenine nucleotide levels specifically in cardiac tissue

    • Investigate tissue-specific metabolic adaptations to altered mitochondrial dynamics

    • Explore connections to heart failure progression and therapeutic interventions

• Therapeutic target validation:

  • Drug screening platforms:

    • Use DNM1L (Ab-637) Antibody to monitor effects of compounds on DNM1L activity

    • Mdivi-1 and related compounds have shown therapeutic potential in models of PAH

    • Evaluate metabolic reprogramming induced by DNM1L-targeting therapies

  • Gene therapy approaches:

    • Assess viral delivery of DNM1L modulators in disease models

    • Monitor effects on mitochondrial function and broader cellular metabolism

    • Correlate therapeutic efficacy with changes in metabolic parameters

By integrating these methodological approaches, researchers can establish causal relationships between DNM1L-mediated mitochondrial dynamics and broader cellular metabolic reprogramming in disease states, potentially identifying new therapeutic targets and biomarkers.