D14 Antibody

What is D14 and what cellular functions is it associated with?

D14, also known as Differentiation related gene 14 protein (with synonyms including ACHP, C7orf2, DIF14), is a protein encoded by the LMBR1 gene . The protein is expressed in human, mouse, and rat tissues, making it a conserved target across these mammalian models . While the search results don't provide explicit details on its cellular functions, the availability of antibodies against D14 across multiple species indicates its research significance in comparative biology studies. When designing experiments to investigate D14 function, researchers should consider its cross-species conservation and potential role in differentiation processes as suggested by its nomenclature.

What detection methods are most effective for D14 protein studies?

Based on validated applications for commercial D14 antibodies, Western blotting (WB) represents the most consistently supported detection method for D14 protein studies . Immunofluorescence on paraffin-embedded sections (IF-p) is also a validated application . When designing experimental workflows:

• Western blotting provides quantitative analysis of D14 expression levels and protein size verification

• Immunofluorescence enables spatial localization studies within tissues or cells

• For IF applications, dilution ratios of 1:50-1:200 have been validated for optimal signal-to-noise ratio

Researchers should incorporate appropriate positive and negative controls when establishing these techniques for D14 detection in their specific experimental systems.

How should D14 antibodies be stored and handled to maintain optimal activity?

Proper storage and handling of D14 antibodies is critical for maintaining their specificity and sensitivity in experimental applications. The recommended storage protocol includes:

• Store at -20°C in the original buffer solution containing glycerol (50%), TBS (0.01M, pH 7.4), BSA (1%), and preservative (ProClin)

• Aliquot into multiple vials upon receipt to avoid repeated freeze-thaw cycles that can denature antibody proteins

• Expected shelf life is approximately 12 months when stored properly

• Allow antibody to reach room temperature before opening for use

For dilution protocols, researchers should prepare working solutions in appropriate buffers immediately before use rather than storing diluted antibody for extended periods, as this can compromise detection sensitivity.

What are the recommended positive controls for D14 antibody validation?

When validating D14 antibodies for experimental use, researchers should include appropriate positive controls based on the target species. While the search results don't specify particular positive control samples, the antibody's validated reactivity to human, mouse, and rat samples suggests using lysates or tissues from these species with known D14 expression . Commercially available recombinant D14 protein could serve as a defined positive control for initial antibody characterization. Antibody validation should include:

• Side-by-side comparison with alternative D14 antibody clones where available

• Blocking peptide experiments using the immunogen (KLH-conjugated synthetic peptide derived from human LMBR1/DIF14)

• Genetic controls (knockout/knockdown) where feasible to confirm specificity

How can fluorophore-conjugated D14 antibodies be optimized for multiplexed imaging applications?

The availability of D14 antibodies conjugated to multiple fluorophores (AbBy Fluor® 350, 488, 555, 594, 647, 680, and 750) enables sophisticated multiplexed imaging applications. When designing multiplexed experiments:

• Select complementary fluorophores with minimal spectral overlap based on your imaging system's filter sets

• Consider the following conjugate properties for experimental design:

When implementing multiplexed protocols, researchers should:

• Conduct single-color controls to assess and correct for spectral overlap

• Adjust antibody concentrations individually for each fluorophore variant as conjugation efficiency may vary

• Consider sequential rather than simultaneous staining when cross-reactivity is observed

What approaches can resolve inconsistent D14 antibody staining patterns across different tissue preparations?

Researchers encountering variable D14 staining patterns across different tissue preparations should systematically evaluate several parameters:

• Fixation effects: The polyclonal D14 antibodies have been validated for paraffin-embedded sections , but epitope masking may occur with certain fixatives. Test multiple fixation protocols (4% PFA, methanol, acetone) to optimize epitope accessibility.

• Antigen retrieval: For formalin-fixed samples, compare:

  • Heat-induced epitope retrieval (citrate buffer pH 6.0 vs. EDTA buffer pH 9.0)

  • Enzymatic retrieval (proteinase K, trypsin)

  • Determine optimal retrieval duration (5-30 minutes)

• Antibody concentration titration: The recommended dilution range for IF applications (1:50-1:200) provides a starting point, but systematic titration specific to your tissue type may be necessary.

• Signal amplification: For low-abundance targets, consider:

  • Tyramide signal amplification

  • Secondary antibody layering techniques

  • Enhanced detection systems (streptavidin-biotin)

Document all optimization steps methodically to establish a reproducible protocol for D14 detection across different tissue preparations.

How can researchers distinguish between specific and non-specific binding when using D14 antibodies?

Distinguishing specific from non-specific binding represents a critical validation step for D14 antibody-based studies. Implement the following comprehensive approach:

• Blocking optimization: Systematically test:

  • Different blocking agents (BSA, normal serum, casein, commercial blockers)

  • Blocking concentrations (1-5%)

  • Blocking duration (30 minutes to overnight)

• Critical controls:

  • Pre-adsorption with immunizing peptide (derived from human LMBR1/DIF14)

  • Secondary-only controls (omitting primary antibody)

  • Isotype controls (irrelevant rabbit IgG at matching concentration)

  • Cell/tissue negative controls (samples with confirmed absence of D14 expression)

• Signal validation techniques:

  • RNA-protein correlation (compare antibody staining with mRNA expression data)

  • siRNA knockdown validation

  • Orthogonal detection methods (alternative antibody clones)

• For Western blotting applications:

  • Include molecular weight markers to confirm band size

  • Evaluate background across the entire membrane

  • Test different blocking/washing stringency conditions

What methodological considerations should be addressed when using D14 antibodies for quantitative protein expression analysis?

For accurate quantitative analysis of D14 protein expression using the available antibodies, researchers should address several methodological challenges:

• Standard curve development:

  • Use recombinant D14 protein at known concentrations

  • Establish linear detection range for both Western blotting and immunofluorescence applications

  • Document lower limit of detection and saturation threshold

• Normalization strategy:

  • For Western blotting: Test multiple loading controls (β-actin, GAPDH, tubulin)

  • For immunofluorescence: Implement consistent exposure settings and internal calibration controls

• Technical replication requirements:

  • Minimum triplicate technical replicates

  • Independent biological replicates (n ≥ 3)

  • Statistical power analysis to determine appropriate sample size

• Image analysis for immunofluorescence quantification:

  • Establish consistent thresholding parameters

  • Use automated quantification algorithms to reduce operator bias

  • Document all image acquisition parameters (exposure, gain, offset)

• Batch effects mitigation:

  • Process all comparative samples simultaneously when possible

  • Include internal reference samples across multiple experiments

  • Apply appropriate statistical corrections for batch variation

How can researchers resolve high background issues when using D14 antibodies?

High background signal represents a common challenge when implementing D14 antibody protocols. A systematic troubleshooting approach includes:

• Antibody concentration optimization:

  • Conduct serial dilution testing beyond the recommended 1:50-1:200 range

  • Test concentrations in 2-fold increments to identify optimal signal-to-noise ratio

• Buffer composition modifications:

  • Add detergents (0.1-0.3% Triton X-100, 0.05-0.1% Tween-20)

  • Increase salt concentration (150-500 mM NaCl)

  • Add carrier proteins (0.1-1% BSA)

• Washing protocol enhancements:

  • Increase wash duration (5-15 minutes per wash)

  • Add additional wash steps (3-5 washes)

  • Test different washing buffers (PBS vs. TBS)

• Tissue-specific considerations:

  • Evaluate autofluorescence reduction techniques for IF applications

  • Consider tissue clearing methods for thick sections

  • Test different blocking agents specific to tissue type

• Storage-related issues:

  • Verify antibody hasn't undergone excessive freeze-thaw cycles

  • Check for signs of microbial contamination

  • Confirm antibody hasn't exceeded 12-month shelf life

What experimental design considerations are critical when comparing D14 expression across different species?

• Epitope conservation analysis:

  • Conduct sequence alignment of the immunogen region across target species

  • Document percent identity and potential species-specific post-translational modifications

  • Validate antibody performance separately for each species

• Standardized sample preparation:

  • Implement identical fixation and processing protocols across species

  • Standardize tissue collection timepoints relative to developmental stage rather than absolute age

  • Account for tissue architecture differences when interpreting localization patterns

• Quantification considerations:

  • Establish species-specific baseline expression levels

  • Verify linear detection range independently for each species

  • Apply appropriate normalization strategies that account for species differences

• Experimental controls:

  • Include positive and negative tissue controls from each species in the same experimental run

  • Validate antibody specificity independently for each species

  • Consider using multiple antibodies targeting different D14 epitopes to confirm patterns