Recombinant Mouse Protein DDX26B (Ddx26b), partial

Basic Research Questions

• What is Recombinant Mouse Protein DDX26B and what are its key structural characteristics?

DDX26B (DEAD/H (Asp-Glu-Ala-Asp/His) box polypeptide 26B) is also known as Integrator complex subunit 6-like (Ints6l). The protein is identified by several alternative gene names including D130066O12, 4930535D10Rik, and 6330505F04Rik . It belongs to the DEAD-box protein family, suggesting potential roles in RNA metabolism, although its specific functions are still being investigated.

The full-length mouse DDX26B protein consists of 861 amino acids . Structurally, it contains functional domains characteristic of DEAD-box proteins, including RNA-binding motifs and potential helicase activity regions. When produced as a recombinant protein, DDX26B is typically available in liquid or lyophilized powder form, with a documented purity level of >80% as determined by SDS-PAGE .

Key physical and biochemical properties include:

• Molecular weight: Approximately 97 kDa

• Endotoxin level: < 1.0 EU per μg of protein (LAL method)

• Storage buffer: PBS

• Stability: Short-term storage at +4°C; long-term storage at -20°C to -80°C

• What expression systems are optimal for producing functional Recombinant Mouse DDX26B?

The expression system selection significantly impacts the structural integrity and functionality of the recombinant protein. Based on current research methodologies, several systems are employed with varying advantages:

For experiments requiring authentic protein function, mammalian expression systems are generally preferable as they provide proper folding and post-translational modifications that more closely resemble the native protein . The purification of DDX26B typically involves affinity chromatography, with one-step protocols being common for tagged versions of the protein .

• How do different purification tags affect DDX26B functionality in experimental applications?

The choice of purification tag can significantly impact experimental outcomes when working with Recombinant Mouse DDX26B. Understanding these effects is crucial for experimental design:

His(Fc)-Avi-tag:
This more complex tagging system combines the purification advantages of the His-tag with the stability benefits of an Fc fragment and the biotinylation capabilities of an Avi-tag . This combination can be particularly useful for complex experimental applications like pull-down assays or surface immobilization, but the larger tag size may have more pronounced effects on protein structure.

Methodological considerations for tag selection:

• Location matters: N-terminal versus C-terminal tagging may differently affect protein folding and function

• Experimental validation: Always compare tagged protein activity with known benchmarks when possible

• Cleavage options: Consider incorporating protease recognition sites if tag removal is necessary for downstream applications

• Control experiments: Include appropriate controls to assess potential tag interference with protein function

When designing experiments, researchers should select the tag system that best balances purification efficiency with minimal functional interference based on their specific research questions.

• What are the recommended experimental conditions for handling and storing Recombinant Mouse DDX26B?

Proper handling and storage of Recombinant Mouse DDX26B are critical for maintaining protein integrity and experimental reproducibility. Based on established protocols, the following methodological guidelines should be followed:

Storage Conditions:

• Short-term storage (up to 2 weeks): +4°C in PBS buffer

• Long-term storage: -20°C to -80°C, preferably in small aliquots to avoid repeated freeze-thaw cycles

• If supplied as lyophilized powder, reconstitute immediately before use or store the powder at -20°C

Working Conditions:

• Temperature: Perform experiments at controlled temperatures (typically 4°C for binding studies or room temperature for activity assays)

• Buffer composition: PBS is the standard storage buffer , but experimental buffers should be optimized based on the specific application

• pH range: Typically 7.2-7.4 for maximum stability

• Avoid repeated freeze-thaw cycles, which can lead to protein denaturation and aggregation

Stability Assessment:

• Monitor protein stability using analytical techniques such as dynamic light scattering or size-exclusion chromatography

• For long-term studies, verify protein activity at regular intervals

• Consider adding stabilizing agents such as glycerol (5-10%) for freeze-thaw protection

By adhering to these handling conditions, researchers can ensure that their experimental results reflect the true properties of DDX26B rather than artifacts from improper storage or handling.

• What functional assays are appropriate for characterizing Recombinant Mouse DDX26B activity?

Given that DDX26B is a member of the DEAD/H box family of proteins, several functional assays can be employed to characterize its biochemical activities. The selection of appropriate assays should be guided by the specific research questions:

RNA-Related Activity Assays:

• RNA binding assays: Electrophoretic mobility shift assays (EMSA) or filter binding assays to assess RNA binding specificity and affinity

• ATP hydrolysis assays: Colorimetric or radiometric assays to measure ATPase activity, which is often coupled to RNA binding in DEAD-box proteins

• RNA duplex unwinding assays: Fluorescence-based methods to assess potential helicase activity

Protein Interaction Assays:

• Pull-down assays using tagged DDX26B to identify interaction partners

• Surface plasmon resonance (SPR) for quantitative measurement of binding kinetics

• Co-immunoprecipitation followed by Western blotting using specific antibodies

Cell-Based Functional Assays:

• Localization studies using immunofluorescence to determine subcellular distribution

• Gene expression analysis following DDX26B overexpression or knockdown

• RNA processing or stability assays to assess potential roles in RNA metabolism

When designing these functional assays, researchers should include appropriate positive and negative controls, optimize reaction conditions, and ensure that the recombinant protein preparation maintains its native structure and activity.

Advanced Research Questions

• How does partial versus full-length DDX26B impact experimental outcomes in interaction studies?

The decision to use partial or full-length Recombinant Mouse DDX26B significantly influences experimental design and interpretation in interaction studies. This methodological consideration requires careful analysis:

Structural and Functional Implications:

Methodological Strategies for Interaction Studies:

• Complementary approach: Begin with partial constructs for initial screening, then validate with full-length protein

• Domain mapping: Use systematically designed partial constructs to identify specific interaction regions

• Structural context: Consider how domain truncation might affect three-dimensional structure and binding interfaces

• Control experiments: Include experiments to verify that interactions observed with partial constructs occur with full-length protein

• What experimental design considerations are critical when studying DDX26B's role in cellular pathways?

Designing robust experiments to elucidate DDX26B's biological functions requires systematic planning following established experimental design principles . Key methodological considerations include:

Independent Variable Manipulation:

• Overexpression approaches: Transient versus stable expression; inducible systems; expression level control

• Loss-of-function approaches: siRNA knockdown; CRISPR-Cas9 knockout; dominant-negative constructs

• Rescue experiments: Re-expression of wild-type or mutant DDX26B in knockout backgrounds

Dependent Variable Selection and Measurement:

• Molecular readouts: Transcriptome analysis; proteomics; RNA processing metrics

• Cellular phenotypes: Proliferation; differentiation; stress response; cell cycle progression

• Biochemical activities: RNA binding; ATP hydrolysis; protein complex formation

Control of Extraneous Variables:

• Genetic background standardization

• Environmental condition consistency (temperature, media composition, cell density)

• Temporal considerations (expression duration, cell cycle synchronization)

Experimental Controls:

• Positive controls: Related DEAD-box proteins with known functions

• Negative controls: Non-functional DDX26B mutants; unrelated proteins of similar size

• Vehicle controls: Empty vector; scrambled siRNA

Statistical Design Considerations:

• Sample size determination through power analysis

• Randomization and blinding where applicable

• Appropriate statistical tests based on data distribution

• Multiple testing correction for high-throughput data

By incorporating these experimental design principles, researchers can generate reliable and reproducible data on DDX26B's cellular functions, minimizing confounding factors and strengthening causal inferences.

• How can researchers resolve contradictory data when characterizing DDX26B's molecular functions?

When faced with contradictory results regarding DDX26B's functions, a systematic troubleshooting approach is necessary. This methodological framework helps reconcile disparate findings:

Data Reconciliation Strategy:

• Technical Variation Assessment:

  • Compare experimental protocols in detail, identifying subtle differences in:

    • Protein preparation (expression system, purification method, tag position)

    • Assay conditions (buffer composition, temperature, incubation time)

    • Detection methods (antibody specificity, detection limits, signal-to-noise ratio)

  • Replicate experiments using standardized protocols to determine reproducibility

• Biological Context Evaluation:

  • Cell type specificity: DDX26B may function differently across cell types

  • Developmental stage variation: Protein function may change throughout development

  • Stress conditions: Function may be altered under different cellular stresses

  • Protein complex composition: Different interaction partners may modulate function

• Construct-Specific Differences:

  • Compare amino acid boundaries of partial constructs used

  • Assess potential effects of different tags (His-tag vs. His(Fc)-Avi-tag )

  • Consider post-translational modifications present in different expression systems

• Isoform and Homology Analysis:

  • Check if contradictory results stem from different isoforms

  • Consider potential confusion between DDX26B and its paralog INTS6

  • Review species differences (mouse vs. human DDX26B)

• Integrative Analysis Approach:

  • Triangulate findings using multiple complementary techniques

  • Weight evidence based on methodological rigor

  • Develop testable hypotheses to specifically address contradictions

When publishing results, researchers should explicitly discuss contradictory findings in the literature and provide possible explanations for discrepancies, advancing the collective understanding of DDX26B functions.

• What troubleshooting approaches are effective for optimizing DDX26B protein expression and purification?

Optimizing DDX26B expression and purification requires systematic troubleshooting to address common challenges:

Expression Optimization:

Purification Optimization:

Quality Control Tests:

• SDS-PAGE analysis targeting >80% purity or ≥85% purity

• Western blot confirmation of identity

• Mass spectrometry for precise molecular weight determination

• Dynamic light scattering for aggregation assessment

• Endotoxin testing using LAL method (target: <1.0 EU per μg)

By systematically addressing these challenges, researchers can optimize DDX26B production while maintaining protein quality and functional integrity.

• How should researchers validate antibody specificity when studying DDX26B in complex biological samples?

Rigorous antibody validation is essential for obtaining reliable data in DDX26B research. The following methodological framework ensures antibody specificity:

Multi-level Validation Strategy:

• Expression-based Validation:

  • Overexpression controls: Test antibody against samples with recombinant DDX26B expression

  • Knockout/knockdown controls: Verify signal reduction in cells lacking DDX26B

  • Cell-type specificity: Test across multiple cell types with varying endogenous expression levels

• Molecular Specificity Validation:

  • Western blot: Confirm single band at expected molecular weight (~97 kDa)

  • Peptide competition: Pre-incubate antibody with immunizing peptide (ASTEPEQLGS VPTDESAITQ MCEVTGGRSY CVRTQRMLNQ CLESLVQKVQ)

  • Immunoprecipitation-mass spectrometry: Verify that DDX26B is the primary protein isolated

• Cross-reactivity Assessment:

  • Test against related proteins, particularly INTS6 (important paralog)

  • Evaluate species cross-reactivity based on sequence homology data (Cow: 93%; Dog: 100%; Guinea Pig: 100%; Horse: 100%; Human: 100%; Mouse: 93%; Rabbit: 100%; Rat: 100%; Zebrafish: 100%)

  • Assess non-specific binding to other DEAD/H box family members

• Application-specific Validation:

  • Western blot: Optimize blocking, antibody concentration, and detection method

  • Immunohistochemistry: Include appropriate tissue controls and optimize antigen retrieval

  • Immunofluorescence: Compare subcellular localization with GFP-tagged DDX26B expression

• Batch Consistency Monitoring:

  • Maintain reference samples for inter-lot comparison

  • Document validation data for each antibody lot

  • Consider monoclonal antibodies for higher reproducibility when available

By implementing this comprehensive validation strategy, researchers can ensure their findings accurately reflect DDX26B biology rather than antibody artifacts.

• What are the most effective experimental designs for investigating DDX26B's potential RNA helicase activity?

As a member of the DEAD/H box family, DDX26B may possess RNA helicase activity, though this requires rigorous experimental confirmation. The following methodological approach provides a comprehensive framework for characterizing this potential function:

Step-wise Functional Characterization:

• ATP Binding and Hydrolysis Assays:

  • Colorimetric ATPase assays (Malachite Green) to quantify inorganic phosphate release

  • Radiometric assays using [γ-32P]ATP to measure ATP hydrolysis rates

  • Experimental variables to test:

    • RNA dependence (with/without RNA substrates)

    • Divalent cation requirements (Mg2+, Mn2+)

    • Temperature and pH optima

• RNA Binding Characterization:

  • Electrophoretic mobility shift assays (EMSA) with labeled RNA substrates

  • Filter binding assays for quantitative binding measurements

  • Fluorescence anisotropy to determine binding kinetics

  • Critical controls: Competition with unlabeled RNA; non-specific RNA controls

• Helicase Activity Measurement:

  • Fluorescence-based unwinding assays using dual-labeled RNA duplexes

  • Radiometric helicase assays using 32P-labeled substrates

  • Experimental design considerations:

    • Substrate design (duplex length, stability, overhang requirements)

    • Time-course experiments to determine unwinding rates

    • ATP dependence and nucleotide specificity

• Structure-Function Analysis:

  • Mutational analysis of conserved motifs (particularly the DEAD/H motif)

  • Truncation constructs to identify minimal functional domains

  • Structural studies (X-ray crystallography or cryo-EM) to elucidate mechanistic details

• Cellular Validation:

  • Identification of physiological RNA targets using CLIP-seq

  • Functional assays in cells with wild-type vs. catalytically inactive DDX26B

This comprehensive experimental approach allows for rigorous testing of DDX26B's biochemical activities while controlling for potential confounding factors, providing clear evidence of its molecular function.

• How can researchers design experiments to differentiate between DDX26B and its paralog INTS6 functions?

The functional distinction between DDX26B (also known as INTS6L) and its paralog INTS6 presents a significant challenge in experimental design. The following methodological framework enables clear functional differentiation:

Systematic Differentiation Strategy:

• Expression Pattern Analysis:

  • Tissue-specific expression profiling using qRT-PCR

  • Cell type-specific expression using single-cell RNA-seq data

  • Developmental timing of expression

  • Subcellular localization comparison using specific antibodies or tagged constructs

• Molecular Tools for Specific Targeting:

  • Design highly specific siRNAs targeting non-homologous regions

  • Develop paralog-specific CRISPR-Cas9 knockout strategies

  • Generate antibodies against unique epitopes (avoid conserved domains)

  • Create rescue constructs resistant to knockdown but functionally intact

• Interactome Mapping:

  • Specific immunoprecipitation followed by mass spectrometry

  • BioID or APEX2 proximity labeling with stringent controls

  • Yeast two-hybrid screening with full-length proteins and domain constructs

  • Comparative analysis to identify unique vs. shared interaction partners

• Functional Complementation Testing:

  • Knockout one paralog and test for functional rescue with the other

  • Domain-swapping experiments between paralogs

  • Evolutionary analysis across species with different paralog compositions

• Physiological Context Differentiation:

  • Stress-specific responses (oxidative, genotoxic, heat shock)

  • Cell-cycle dependent functions

  • Developmental stage-specific requirements

Importantly, all experiments should include rigorous controls validating the specificity of reagents used to target each paralog. When interpreting results, researchers should explicitly consider potential functional redundancy versus specialization between DDX26B and INTS6.