DPPA5 Antibody, FITC conjugated

What is DPPA5 and what are its key biological functions?

DPPA5 is a 116 amino acid protein that localizes to the cytoplasm and contains one KH domain. It is primarily expressed in embryonic germ (EG), primordial germ (PG), and embryonic stem (ES) cells . DPPA5 plays a crucial role in the maintenance of embryonic stem cell pluripotency but is dispensable for self-renewal of pluripotent ES cells and establishment of germ cells . Recent research has demonstrated that DPPA5 associates with specific target mRNAs and functions as a post-transcriptional regulator .

Methodologically, researchers investigating DPPA5 function should consider both gain-of-function and loss-of-function approaches using overexpression vectors and RNA interference techniques, respectively. Protein-RNA interaction studies such as RNA immunoprecipitation (RIP) or crosslinking immunoprecipitation (CLIP) are recommended to characterize its RNA binding properties.

What are the main applications for DPPA5 Antibody, FITC conjugated?

DPPA5 Antibody, FITC conjugated can be utilized in multiple immunological detection techniques:

When designing experiments, researchers should always include appropriate positive controls (such as embryonic stem cells or embryonal carcinoma cells) and negative controls (differentiated cells lacking DPPA5 expression). The FITC conjugation eliminates the need for secondary antibody incubation, simplifying protocols and reducing background in multicolor immunofluorescence experiments.

How should DPPA5 Antibody, FITC conjugated be stored and handled?

For optimal performance and longevity, DPPA5 Antibody, FITC conjugated should be stored at -20°C in small aliquots to avoid repeated freeze-thaw cycles . The antibody is typically shipped in a storage buffer containing 0.01M TBS (pH 7.4) with 1% BSA, 0.03% Proclin300, and 50% Glycerol .

When handling the antibody, researchers should:

• Thaw aliquots completely before use and mix gently

• Avoid exposure to light to prevent photobleaching of the FITC fluorophore

• Keep on ice during experimental procedures

• Return unused portions to -20°C promptly

• Monitor for signs of degradation (reduced signal intensity, increased background)

How can DPPA5 Antibody, FITC conjugated be used to investigate pluripotency mechanisms?

Recent research has demonstrated that DPPA5 directly interacts with, stabilizes, and enhances the function of NANOG in human pluripotent stem cells (hPSCs) . To investigate pluripotency mechanisms using DPPA5 Antibody, FITC conjugated, researchers can:

• Perform co-localization studies with other pluripotency factors (OCT4, SOX2, NANOG) using multi-color immunofluorescence

• Track DPPA5 expression dynamics during differentiation or reprogramming processes

• Combine with chromatin immunoprecipitation (ChIP) to investigate genomic binding sites

• Use flow cytometry for quantitative assessment of DPPA5 expression in heterogeneous cell populations

When designing these experiments, it's critical to understand that DPPA5 overexpression increases NANOG protein levels via a post-transcriptional mechanism, without affecting NANOG transcript levels . This suggests examining protein stability rather than transcriptional regulation when studying DPPA5-NANOG interactions.

What are the best strategies for optimizing immunofluorescence protocols with DPPA5 Antibody, FITC conjugated?

Optimizing immunofluorescence protocols with DPPA5 Antibody, FITC conjugated requires attention to several critical parameters:

• Fixation method: 4% paraformaldehyde (10-15 minutes at room temperature) typically preserves both antigenicity and cellular morphology

• Permeabilization: 0.1-0.3% Triton X-100 for 5-10 minutes usually provides adequate access to cytoplasmic DPPA5

• Blocking: 5-10% normal serum from a species unrelated to the primary antibody host for 1 hour

• Antibody concentration: Start with 1:100 dilution and titrate as needed (1:50-1:200 range)

• Incubation time: Overnight at 4°C often yields best signal-to-noise ratio

• Counterstaining: DAPI (1 μg/mL) for nuclear visualization

• Mounting: Anti-fade mounting medium to prevent photobleaching

When imaging, use appropriate filters for FITC (excitation ~495 nm, emission ~520 nm) and adjust exposure settings to prevent photobleaching while capturing sufficient signal.

How can cross-reactivity of DPPA5 Antibody be assessed across different species?

DPPA5 Antibody shows varying degrees of cross-reactivity across species, which is important to validate for comparative studies. According to BLAST analysis, the percent identity varies: Human (100%), Chimpanzee, Gorilla, Gibbon, Monkey, Marmoset, Mouse, Hamster (100%), Rat, Panda, Rabbit (92%), Elephant, Dog (91%), Bat (84%) .

Western blot analysis shows approximately 40% cross-reactivity with recombinant mouse DPPA5 . To properly assess cross-reactivity:

• Perform sequence alignments of the immunogen region across target species

• Run western blots with positive control samples from each species of interest

• Include recombinant DPPA5 proteins as standards when available

• Validate with alternative detection methods (qPCR, mass spectrometry)

• Perform peptide competition assays to confirm specificity

If cross-reactivity is insufficient for your experimental species, consider using alternative antibodies raised against species-specific epitopes.

What controls should be included when using DPPA5 Antibody, FITC conjugated in immunofluorescence studies?

Proper controls are essential for interpreting immunofluorescence results with DPPA5 Antibody, FITC conjugated:

Additional considerations include:

• Testing antibody performance on both fixed and unfixed samples

• Using serial dilutions to determine optimal concentration

• Including untreated cells to assess basal expression levels

• Running parallel western blots to confirm specificity by molecular weight

How can DPPA5 Antibody, FITC conjugated be used in flow cytometry for stem cell characterization?

Flow cytometry with DPPA5 Antibody, FITC conjugated offers quantitative analysis of DPPA5 expression in heterogeneous cell populations. The protocol typically involves:

• Harvesting cells via gentle enzymatic dissociation (TrypLE or Accutase for stem cells)

• Fixing cells with 2-4% paraformaldehyde for 10-15 minutes

• Permeabilizing with 0.1% saponin or 0.1% Triton X-100

• Blocking with 1-5% BSA in PBS for 30 minutes

• Incubating with DPPA5 Antibody, FITC conjugated (titrate from 1:50-1:200)

• Washing thoroughly with PBS containing 0.1% BSA

• Analyzing using appropriate FITC detection channels (488nm excitation laser, 530/30nm bandpass filter)

When designing multicolor panels, consider:

• Compensating for spectral overlap with other fluorophores

• Including viability dyes to exclude dead cells

• Co-staining with other pluripotency markers (OCT4, NANOG, SOX2)

• Using appropriate isotype controls for gating strategy development

This approach enables quantification of DPPA5+ cells during differentiation or reprogramming processes, and can be combined with cell sorting for subsequent molecular or functional analyses.

What approaches can resolve inconsistent staining results with DPPA5 Antibody, FITC conjugated?

Inconsistent staining with DPPA5 Antibody, FITC conjugated may stem from various technical factors. Here's a systematic troubleshooting approach:

• Antibody integrity:

  • Check for signs of degradation (precipitates, color changes)

  • Ensure proper storage conditions (-20°C, protected from light)

  • Validate with fresh aliquots or alternative lot numbers

• Sample preparation:

  • Optimize fixation duration (over-fixation can mask epitopes)

  • Test different permeabilization reagents and concentrations

  • Ensure samples are processed consistently across experiments

• Protocol optimization:

  • Titrate antibody concentration (1:50-1:200)

  • Adjust incubation temperature and duration

  • Modify blocking conditions to reduce background

• Technical considerations:

  • Protect from light during all steps to prevent photobleaching

  • Ensure even reagent distribution across the sample

  • Use freshly prepared buffers and solutions

• Biological variables:

  • Confirm DPPA5 expression in your cell type (NTera-2 cells as positive control)

  • Consider cell cycle effects (DPPA5 expression may fluctuate)

  • Account for culture conditions (feeder-free cultures show higher DPPA5 expression)

How does DPPA5 interact with NANOG to support pluripotency and reprogramming?

Recent research has elucidated a critical mechanism by which DPPA5 supports pluripotency through direct interaction with NANOG . Specifically:

• DPPA5 directly interacts with NANOG protein, as demonstrated by co-immunoprecipitation experiments

• This interaction stabilizes NANOG, increasing its half-life and protein levels without affecting NANOG mRNA levels

• DPPA5 enhances NANOG function in human pluripotent stem cells

• DPPA5 increases reprogramming efficiency of human somatic cells to induced pluripotent stem cells (hiPSCs)

To investigate this interaction using DPPA5 Antibody, FITC conjugated:

• Perform co-localization studies with NANOG antibodies using confocal microscopy

• Combine with proximity ligation assays to visualize direct protein-protein interactions

• Use fluorescence resonance energy transfer (FRET) to analyze the spatial relationship between DPPA5 and NANOG

• Employ immunoprecipitation followed by mass spectrometry to identify additional interaction partners

This research has significant implications for optimizing reprogramming protocols and understanding the regulatory networks governing pluripotency.

How does culture substrate affect DPPA5 expression in human pluripotent stem cells?

Culture conditions significantly impact DPPA5 expression levels in human pluripotent stem cells (hPSCs). Specifically, cells cultured on feeder-free substrates demonstrate higher DPPA5 gene expression and protein levels compared to those grown on mouse embryonic fibroblasts (MEFs) . Various feeder-free substrates associated with elevated DPPA5 expression include:

• Matrigel

• Laminin-511

• Vitronectin

• Synthetic polymers such as poly[2-(methacryloyloxy) ethyl dimethyl-(3-sulfopropyl) ammonium hydroxide]

Researchers investigating DPPA5 should:

• Consider culture substrate as a critical variable in experimental design

• Monitor DPPA5 expression when transitioning between culture systems

• Standardize culture conditions when comparing DPPA5 levels across experimental groups

• Use DPPA5 Antibody, FITC conjugated to quantify expression differences via flow cytometry or immunofluorescence

These findings suggest that DPPA5 may be part of a mechanistic pathway through which culture substrates influence pluripotency maintenance, which warrants further investigation.

What methodological approaches can characterize DPPA5's role in RNA regulation in stem cells?

DPPA5 contains a KH domain , suggesting it functions as an RNA-binding protein. To investigate its role in RNA regulation using DPPA5 Antibody, FITC conjugated alongside other methodologies:

• RNA-protein interaction studies:

  • RNA immunoprecipitation (RIP) using DPPA5 antibodies followed by RNA-seq

  • CLIP-seq (Crosslinking immunoprecipitation) to identify direct RNA binding sites

  • Biotinylated RNA pull-down assays to confirm specific interactions

• Functional analyses:

  • DPPA5 knockdown/knockout followed by transcriptome analysis

  • Polysome profiling to assess effects on translation

  • RNA stability assays using actinomycin D chase experiments

• Cellular localization:

  • Co-staining with RNA granule markers (P-bodies, stress granules)

  • Subcellular fractionation followed by western blotting

  • FISH (Fluorescence in situ hybridization) combined with DPPA5 immunofluorescence

• Structure-function studies:

  • Mutagenesis of the KH domain to identify critical residues

  • Domain deletion analysis to map regions required for RNA binding

  • Recombinant protein production for in vitro binding assays

These approaches can elucidate how DPPA5 regulates specific mRNAs to maintain pluripotency and facilitate cellular reprogramming.

How do different DPPA5 antibody conjugates compare in research applications?

Various DPPA5 antibody conjugates offer distinct advantages for different experimental approaches:

When selecting between conjugates, researchers should consider:

• The specific detection method and instrumentation available

• Requirements for multi-parameter analysis (fluorescent conjugates for multi-color experiments)

• Sensitivity requirements (enzymatic conjugates for signal amplification)

• Protocol complexity preferences (direct conjugates for simpler workflows)

FITC-conjugated DPPA5 antibodies excel in applications requiring direct visualization or quantification of DPPA5 expression, particularly in multi-color immunofluorescence or flow cytometry experiments .

How can contradictory DPPA5 expression data be reconciled across different experimental systems?

Researchers may encounter contradictory data regarding DPPA5 expression across different experimental systems. These discrepancies may arise from:

• Technical variables:

  • Antibody specificity and sensitivity differences

  • Detection method sensitivity thresholds

  • Sample preparation variations (fixation, permeabilization)

  • Detection of different DPPA5 isoforms or post-translational modifications

• Biological variables:

  • Culture conditions (feeder vs. feeder-free systems)

  • Passage number and cellular heterogeneity

  • Cell cycle phase variation (if DPPA5 expression is cell-cycle dependent)

  • Species differences (human vs. mouse models)

To reconcile contradictory data:

• Validate findings using multiple antibodies targeting different epitopes

• Confirm antibody specificity through knockout/knockdown controls

• Complement protein detection with mRNA analysis (RT-qPCR, RNA-seq)

• Standardize experimental conditions across studies

• Consider quantitative methods (flow cytometry, western blot with densitometry) over qualitative assessment

Understanding the context-dependent regulation of DPPA5 expression can provide insights into its role in pluripotency networks across different model systems.