DYNLL1 Recombinant Monoclonal Antibody

What is DYNLL1 and why is it an important research target?

DYNLL1 (Dynein Light Chain 1, also known as LC8) functions as a non-catalytic accessory component of the cytoplasmic dynein 1 complex involved in linking dynein to cargos and adapter proteins that regulate dynein function. Beyond its role in cytoskeletal transport, DYNLL1 serves as a protein-protein adapter that inhibits and/or sequesters target proteins . It plays crucial roles in DNA damage response, immune signaling through the NF-κB pathway, and neuronal function via nitric oxide signaling . Its diverse functions make it an important target for understanding various cellular processes and disease mechanisms.

What are the typical applications for DYNLL1 monoclonal antibodies?

DYNLL1 monoclonal antibodies are commonly used in:

• Western blotting (WB) for protein detection and quantification

• Immunoprecipitation (IP) for protein-protein interaction studies

• Immunofluorescence (IF) and immunocytochemistry (ICC) for subcellular localization

• Flow cytometry (FC) for intracellular detection

• Immunohistochemistry (IHC) for tissue expression analysis

These applications enable researchers to study DYNLL1's expression patterns, interactions, and functions in different experimental contexts.

What is the molecular weight of DYNLL1 and what band size should be expected in Western blots?

The canonical human DYNLL1 protein consists of 89 amino acid residues with a calculated molecular weight of approximately 10.4 kDa . In Western blot applications, DYNLL1 is typically observed at 8-10 kDa . Variations in observed molecular weight may occur due to post-translational modifications or differences in gel systems.

How should I optimize immunoblotting conditions for DYNLL1 detection?

For optimal DYNLL1 detection by Western blotting:

• Sample preparation:

  • Use RIPA or NP-40 based lysis buffers with protease inhibitors

  • Include phosphatase inhibitors if studying phosphorylation states (e.g., phospho-Ser88)

• Gel electrophoresis:

  • Use high percentage (15-18%) SDS-PAGE gels for better resolution of low molecular weight proteins

  • Consider gradient gels (4-20%) if analyzing DYNLL1 complexes

• Transfer conditions:

  • Optimize for small proteins with 100% methanol in transfer buffer

  • Consider semi-dry transfer systems with 20V for 30-40 minutes

• Antibody dilution:

  • Primary antibody dilution typically 1:1000-1:4000

  • Secondary antibody at 1:5000-1:10000

• Signal detection:

  • ECL-based detection systems work well for standard applications

  • Consider fluorescent detection for quantitative analysis

Always validate with positive controls (e.g., HEK-293, HeLa, Jurkat or MCF-7 cells) as these have detectable endogenous DYNLL1 levels .

What are the recommended fixation and permeabilization methods for DYNLL1 immunofluorescence staining?

For optimal immunofluorescence detection of DYNLL1:

• Fixation options:

  • 4% paraformaldehyde (PFA) for 10-15 minutes at room temperature preserves most cellular structures

  • Methanol fixation (-20°C for 10 minutes) may better expose some epitopes and simultaneously permeabilizes the membrane

• Permeabilization (if using PFA):

  • 0.1-0.5% Triton X-100 for 5-10 minutes

  • 0.1% saponin may be preferable for maintaining membrane integrity

• Blocking:

  • 5% normal serum (species of secondary antibody) with 1% BSA

  • 1-2 hour incubation at room temperature

• Antibody dilution:

  • Primary anti-DYNLL1 antibody at 1:200-1:800

  • Incubate overnight at 4°C for best signal-to-noise ratio

• Controls:

  • Include a DYNLL1 knockdown control to verify specificity

  • Use tissue/cells known to express DYNLL1 (e.g., MCF-7 cells)

When studying DYNLL1 in the context of DNA damage response, consider co-staining with DSB markers (e.g., γH2AX) and/or DNA damage response proteins (e.g., 53BP1, MRE11).

How can I differentiate between DYNLL1 and its closely related isoform DYNLL2 in my experiments?

Distinguishing between DYNLL1 and DYNLL2 requires careful experimental design due to their high sequence similarity (93% identity) :

• Antibody selection:

  • Use antibodies raised against unique regions or confirmed by knockout validation

  • Verify specificity using recombinant DYNLL1 and DYNLL2 proteins in Western blots

• siRNA/shRNA approach:

  • Design isoform-specific siRNAs targeting unique regions

  • Validate knockdown specificity by qRT-PCR with isoform-specific primers

  • Confirm protein reduction by Western blot

• CRISPR-Cas9 gene editing:

  • Generate DYNLL1 and DYNLL2 knockout cell lines as definitive controls

  • Use these lines to validate antibody specificity

• Expression analysis:

  • DYNLL1 shows higher expression in testis and moderate levels in brain

  • DYNLL2 has a distinct tissue expression pattern

• Functional assays:

  • DYNLL1 and DYNLL2 have different binding specificities (e.g., DYNLL2 has unique interaction with myosin Va)

  • Design functional assays based on known isoform-specific interactions

What are the recommended approaches for studying DYNLL1 phosphorylation states in the context of DNA damage response?

DYNLL1 phosphorylation, particularly at Ser88, is critical for its function in DNA damage response . To study this:

• Phospho-specific antibodies:

  • Use antibodies specifically recognizing phospho-Ser88 DYNLL1

  • Validate with phosphatase treatment as a negative control

  • Use DYNLL1-S88A mutant expressing cells as additional controls

• Phosphorylation induction:

  • DNA damage can be induced using:

    • Ionizing radiation (5 Gy is commonly used)

    • Radiomimetic drugs (e.g., bleomycin, neocarzinostatin)

    • Laser microirradiation for localized damage

• Temporal dynamics:

  • Perform time-course experiments after DNA damage induction

  • DYNLL1 localizes to DNA lesions rapidly within minutes

• Kinase inhibition studies:

  • Identify responsible kinases using specific inhibitors

  • Confirm with kinase knockdown/knockout approaches

• Protein-protein interactions:

  • Compare interactions of phosphorylated vs. non-phosphorylated DYNLL1

  • Use phosphomimetic (S88D) and phospho-dead (S88A) mutants

  • Phosphorylation state affects DYNLL1's interaction with MRE11, influencing DNA end resection

• Chromatin fractionation:

  • Isolate chromatin after DNA damage to assess DYNLL1 recruitment

  • Compare wild-type and phospho-mutant recruitment dynamics

How can I investigate the role of DYNLL1 in the NF-κB signaling pathway?

DYNLL1 plays a critical role in TLR4-mediated NF-κB pathway activation. To study this function:

• Cellular models:

  • B cells and fibroblasts are suitable models for studying DYNLL1 in NF-κB signaling

  • Generate DYNLL1 knockout/knockdown cells using CRISPR-Cas9 or RNAi

• Pathway activation:

  • Stimulate cells with LPS (TLR4 ligand), IL-1, or CD40 ligand

  • Monitor activation kinetics using time-course experiments

• Readouts for NF-κB activation:

  • IκBα degradation by Western blot

  • IKK phosphorylation/activation assays

  • NF-κB nuclear translocation by immunofluorescence

  • NF-κB DNA binding by EMSA or ChIP

  • NF-κB-dependent gene expression by qRT-PCR or reporter assays

• Rescue experiments:

  • Re-express DYNLL1 in knockout cells to confirm specificity

  • Use domain mutants to identify regions required for NF-κB regulation

• Protein interaction studies:

  • Investigate DYNLL1 interactions with components of the NF-κB pathway

  • Use co-immunoprecipitation followed by Western blot or mass spectrometry

• In vivo validation:

  • Use mouse models with B-cell-specific DYNLL1 deletion

  • Assess immune responses to TLR4-dependent antigens like NP-LPS

How do I resolve contradictory data regarding DYNLL1's role in NF-κB signaling?

Previous studies suggested that overexpressed DYNLL1 inhibits NF-κB activation by blocking IκBα degradation, while newer research indicates DYNLL1 is required for NF-κB activation upstream of IκBα . To resolve such contradictions:

• Expression level considerations:

  • Overexpression artifacts may cause non-physiological effects

  • Compare physiological expression with overexpression systems

  • Use inducible expression systems to create dose-response curves

• Cell type specificity:

  • Test multiple cell types (e.g., B cells, fibroblasts, epithelial cells)

  • Consider tissue-specific functions and interacting partners

• Signal specificity:

  • DYNLL1 is required for TLR4, IL-1, and CD40 signaling but dispensable for TNF-α and BCR signaling

  • Compare multiple stimuli in the same experimental system

• Temporal dynamics:

  • Perform detailed time-course experiments after stimulation

  • Early vs. late effects may differ significantly

• Genetic approaches:

  • Use genetic knockouts rather than knockdowns when possible

  • Create rescue systems with wildtype and mutant DYNLL1

• Analysis pipeline:

  • Examine multiple steps in the NF-κB pathway (IKK activation, IκBα degradation, p65 phosphorylation, nuclear translocation)

  • This helps determine where DYNLL1 acts in the pathway

The current consensus is that at physiological levels, DYNLL1 promotes NF-κB activation in response to specific signals, despite earlier overexpression studies suggesting the opposite .

What controls should be included when validating DYNLL1 monoclonal antibody specificity?

Comprehensive validation of DYNLL1 antibody specificity requires several controls:

• Positive expression controls:

  • Cell lines with confirmed DYNLL1 expression (HEK-293, HeLa, Jurkat, MCF-7)

  • Tissues with high DYNLL1 expression (testis, brain)

• Negative controls:

  • DYNLL1 knockout cells generated by CRISPR-Cas9

  • siRNA/shRNA-mediated DYNLL1 knockdown

  • Peptide competition assays to block specific binding

• Recombinant protein controls:

  • Purified recombinant DYNLL1 protein as positive control

  • Closely related proteins (e.g., DYNLL2) to test cross-reactivity

• Species cross-reactivity:

  • If the antibody claims cross-reactivity with multiple species, test each one

  • Human, mouse, and rat DYNLL1 can be detected with many antibodies

• Application-specific controls:

  • For WB: Molecular weight markers, loading controls

  • For IF/IHC: Secondary antibody-only controls, isotype controls

  • For IP: IgG control, input samples

• Special considerations:

  • Test antibody performance in different experimental conditions

  • Validate under both native and denatured conditions

  • If studying phosphorylated forms, include phosphatase-treated samples

What are the best approaches to analyze DYNLL1's diverse functional roles in different cellular compartments?

DYNLL1 functions in multiple cellular compartments including the cytoplasm, nucleus, and mitochondria. To comprehensively analyze its diverse roles:

• Subcellular fractionation:

  • Isolate nuclear, cytoplasmic, mitochondrial, and chromatin fractions

  • Analyze DYNLL1 distribution by Western blot with compartment-specific markers

  • Monitor redistribution upon various stimuli (e.g., DNA damage, LPS treatment)

• Live-cell imaging:

  • Generate cells expressing fluorescently-tagged DYNLL1 (e.g., EGFP-DYNLL1)

  • Use photobleaching techniques (FRAP/FLIP) to study dynamics

  • Employ super-resolution microscopy for detailed localization

  • Use microirradiation to study recruitment to DNA damage sites

• Proximity labeling techniques:

  • BioID or TurboID fused to DYNLL1 to identify compartment-specific interactors

  • APEX2 for electron microscopy-compatible proximity labeling

  • Analyze data with subcellular annotation to identify compartment-specific functions

• Immunoprecipitation-based approaches:

  • Perform IPs from different subcellular fractions

  • Use crosslinking to capture transient interactions

  • Analyze by mass spectrometry to identify interactors

  • Compare interactomes under different conditions

• Functional assays by compartment:

  • Cytoplasm: Dynein-dependent transport assays, microtubule co-sedimentation

  • Nucleus: DNA damage response, repair pathway choice analysis

  • Mitochondria: Apoptosis assays, interaction with BCL2L11 (BIM)

• Multicolor imaging:

  • Co-stain with markers for different cellular processes

  • DNA repair (53BP1, γH2AX, MRE11)

  • Transport (dynein components)

  • NF-κB signaling (p65, IκBα)

How can I investigate the dynamics of DYNLL1 recruitment to DNA double-strand breaks?

To study DYNLL1 recruitment to DSBs in detail:

• Real-time recruitment assays:

  • Express EGFP-tagged DYNLL1 in appropriate cell lines

  • Induce DNA damage using laser microirradiation

  • Monitor recruitment kinetics by live-cell imaging

  • DYNLL1 localizes to DNA lesions rapidly within minutes, with kinetics comparable to 53BP1

• Protein interaction dynamics:

  • Study interactions with key DSB response factors (53BP1, MRE11)

  • Use fluorescence resonance energy transfer (FRET) to detect direct interactions

  • Employ fluorescence correlation spectroscopy (FCS) for binding dynamics

• Structure-function analysis:

  • Generate point mutants or domain deletions (e.g., S88A, S88D)

  • Analyze recruitment of these mutants after damage

  • Compare phosphorylated vs. non-phosphorylated DYNLL1 dynamics

• Super-resolution microscopy:

  • Use techniques like STORM, PALM, or SIM for precise spatial localization

  • Determine whether DYNLL1 colocalizes with specific repair complexes

• ChIP-based approaches:

  • Perform ChIP-seq after inducing site-specific DSBs

  • DYNLL1 ChIP at endonuclease-induced break sites

  • Compare with other repair factors like 53BP1 and MRE11

• Dependency studies:

  • Use cells lacking specific repair factors (53BP1, RIF1, etc.)

  • DYNLL1 recruitment to DSBs is dependent on 53BP1 but independent of RIF1, REV7, and Shieldin complex components

What approaches can be used to investigate the regulatory relationship between DYNLL1 and the ASCIZ transcription factor?

ASCIZ is the designated transcription factor for DYNLL1. To study this regulatory relationship:

• Transcriptional regulation analysis:

  • Luciferase reporter assays with DYNLL1 promoter constructs

  • ChIP assays to detect ASCIZ binding to the DYNLL1 promoter

  • EMSA to study direct DNA binding by ASCIZ

• Expression correlation studies:

  • Manipulate ASCIZ levels and monitor DYNLL1 expression

  • Analyze correlation in different tissues and cell types

  • Perform qRT-PCR and Western blot analysis

• Feedback regulation:

  • DYNLL1 can regulate ASCIZ transcriptional activity

  • Study how modulating DYNLL1 affects its own transcription

  • Investigate the role of this feedback in different contexts

• Functional outcomes:

  • Compare phenotypes of ASCIZ-deficient and DYNLL1-deficient systems

  • B-cell-specific deletion of either ASCIZ or DYNLL1 leads to similar defects in TLR4-dependent antibody responses

  • Use rescue experiments with exogenous DYNLL1 in ASCIZ-deficient cells

• Stimulus-responsive regulation:

  • Analyze how different stimuli affect the ASCIZ-DYNLL1 axis

  • Study regulation in response to DNA damage, immune activation

  • Monitor dynamics using time-course experiments

What methodological approaches are recommended for studying DYNLL1's role in modulating MRE11 activity during DNA end resection?

DYNLL1 regulates DNA end resection by disrupting MRE11 dimerization. To study this mechanism:

• Biochemical approaches:

  • Gel filtration or analytical ultracentrifugation to assess MRE11 dimerization

  • In vitro nuclease assays with recombinant MRE11 +/- DYNLL1

  • Use purified components to reconstitute the system in vitro

• Structural studies:

  • X-ray crystallography or cryo-EM of DYNLL1-MRE11 complexes

  • Hydrogen-deuterium exchange mass spectrometry to map interaction interfaces

  • Molecular dynamics simulations to understand conformational effects

• Cell-based assays:

  • DNA resection assays (RPA foci, BrdU exposure under native conditions)

  • ssDNA-specific antibody staining after damage

  • Track MRE11 nuclease activity with specialized fluorescent reporters

• Chromatin dynamics:

  • Isolate chromatin after DNA damage induction (e.g., 5 Gy irradiation)

  • Add recombinant DYNLL1 (wild-type or phospho-mutants)

  • Monitor MRE11 displacement from chromatin

• Mutation analysis:

  • Generate MRE11 mutants that disrupt DYNLL1 binding

  • Test their resection activity and dimerization properties

  • Analyze DSB repair pathway choice in cells expressing these mutants

• Single-molecule approaches:

  • Use single-molecule FRET to study MRE11 conformational changes

  • Track individual molecules of MRE11 and DYNLL1 in live cells

  • Perform single-molecule pull-down assays to determine complex stoichiometry

This multi-faceted approach can provide insights into how DYNLL1 mechanistically regulates MRE11 activity to control DNA end resection.