DPPA5 Antibody, Biotin conjugated

What is DPPA5 and why is it significant in stem cell research?

DPPA5 (Developmental Pluripotency Associated 5), also known as ESG1, is a protein that functions in controlling cell pluripotency and early embryogenesis. This 13 kDa cytoplasmic protein is a specific marker for pluripotent stem cells, making it valuable in stem cell research . DPPA5 expression shows a high correlation with POU5F1 (OCT4) expression and other human pluripotent stem cell (hPSC) surface markers such as TRA-160 and SSEA-4 . The protein is particularly important because it can detect rare OCT4-positive cells in differentiated cell cultures and is immunoreactive to cell surface protein epitopes on both primed and naive state human pluripotent stem cells . This makes DPPA5 antibodies essential tools for investigating pluripotency states and cellular reprogramming mechanisms.

What is the advantage of using biotin-conjugated DPPA5 antibodies?

Biotin-conjugated DPPA5 antibodies offer several methodological advantages for researchers:

• Enhanced detection sensitivity: Biotin's high affinity for avidin and streptavidin allows for signal amplification in detection systems.

• Versatile detection strategies: The biotin-conjugated antibody can be detected using various streptavidin-conjugated fluorophores or enzymes, offering flexibility in experimental design.

• Multicolor analysis compatibility: In flow cytometry and immunofluorescence applications, biotin-conjugated antibodies can be paired with directly labeled antibodies for multiparameter analysis.

• Reduced background in complex samples: The biotin-avidin system often provides cleaner results with less non-specific binding in cellular assays .

Research has demonstrated that biotin-conjugated anti-DPPA5 antibodies have been successfully used in ELISA applications for human samples, providing researchers with reliable tools for quantitative protein detection .

What applications are most suitable for biotin-conjugated DPPA5 antibodies?

Based on validated research applications, biotin-conjugated DPPA5 antibodies are particularly well-suited for:

• ELISA: Providing quantitative measurement of DPPA5 protein levels in cell and tissue lysates .

• Immunofluorescence microscopy: For visualizing the cytoplasmic localization of DPPA5 in pluripotent stem cells .

• Flow cytometry: For identifying and sorting DPPA5-expressing cells in heterogeneous populations .

• Co-immunoprecipitation studies: For investigating protein-protein interactions involving DPPA5 .

Research has shown that these antibodies perform consistently across these applications when proper methodological controls are implemented. For example, immunofluorescence studies have successfully used DPPA5 antibodies alongside other pluripotency markers to characterize stem cell populations .

What are the optimal storage and handling conditions for biotin-conjugated DPPA5 antibodies?

For maintaining optimal activity of biotin-conjugated DPPA5 antibodies, researchers should follow these evidence-based handling protocols:

• Storage temperature: Store at -20°C. Long-term storage at this temperature maintains antibody activity for up to 12 months .

• Aliquoting: Upon receipt, divide the antibody into small, single-use aliquots to avoid repeated freeze-thaw cycles, which can degrade the antibody and reduce its specificity .

• Shipping conditions: The antibody is typically shipped with ice packs. Upon receipt, store immediately at the recommended temperature (-20°C) .

• Working solution preparation: Dilute only the amount needed for immediate use in appropriate buffer (typically phosphate-buffered solution with 0.05% stabilizer) .

• Avoid freeze/thaw cycles: Each freeze/thaw cycle can reduce antibody activity by approximately 10-15% .

Following these guidelines ensures maximum antibody performance and reproducibility across experiments.

How can researchers validate the specificity of biotin-conjugated DPPA5 antibodies?

Validation of biotin-conjugated DPPA5 antibodies should follow these methodological steps:

• Western blot analysis: Confirm detection of the expected 13 kDa band in pluripotent stem cell lysates. The observed molecular weight should align with the calculated molecular weight of 13 kDa for DPPA5 .

• Positive and negative controls:

  • Positive: Use cell lines known to express DPPA5 (embryonic stem cells, induced pluripotent stem cells)

  • Negative: Use differentiated cell types that do not express DPPA5

• Cross-reactivity testing: Verify specificity across species if conducting comparative studies. According to antibody specifications, reactivity can vary across species with predictions of 92% for dog, 100% for human and mouse, 79% for pig, 93% for rabbit, and 92% for rat .

• Competition assays: Pre-incubate the antibody with purified DPPA5 protein before application to demonstrate binding specificity.

• Correlation with other pluripotency markers: Verify that DPPA5 detection correlates with OCT4, TRA-160, and SSEA-4 expression patterns in pluripotent stem cells .

What are the recommended dilutions and concentrations for different experimental applications?

Optimal dilutions for biotin-conjugated DPPA5 antibodies vary by application, based on validated research protocols:

The concentration of commercially available biotin-conjugated DPPA5 antibodies is typically 1 mg/mL . Working solutions should be prepared accordingly to achieve optimal signal-to-noise ratios for each specific application. It is recommended to perform a titration experiment when first using the antibody in a new experimental system.

How can DPPA5 antibodies be used to investigate different pluripotency states in human stem cells?

DPPA5 antibodies are valuable tools for distinguishing between naive and primed pluripotency states, as demonstrated by recent research:

• Differential expression patterns: Research has shown that DPPA5 detection using specific antibodies can help identify distinct subpopulations within pluripotent stem cell cultures. DPPA5 antibodies have been used in immunofluorescence analyses to investigate transcriptional heterogeneity in naive versus primed human embryonic stem cells .

• Co-expression analysis: DPPA5 antibodies can be used in conjunction with other markers like ABCG2 to characterize pluripotency states. Imaging studies have demonstrated distinct patterns in naive versus primed hESCs using DPPA5 antibodies .

• Transition monitoring: During the conversion between naive and primed states, DPPA5 antibody staining provides valuable information about the intermediate states. The antibodies are immunoreactive to cell surface protein epitopes on both primed and naive state hPSCs .

• Single-cell analysis: When combined with flow cytometry, DPPA5 antibodies enable researchers to isolate and study subpopulations of cells at different stages of pluripotency, as demonstrated in studies using Smart-seq2 protocol for single-cell transcriptome analysis .

• Culture condition assessment: DPPA5 antibodies have been utilized to evaluate the efficacy of different culture media in maintaining naive pluripotency, including Naive Human Stem Cell Medium (NHSM), 5i/hLIF medium supplemented with FGF and Activin A (5i/L/FA), and RSeT medium .

What methodologies can be employed to study DPPA5's post-translational modifications using specific antibodies?

Research into DPPA5's post-translational modifications, particularly ubiquitination, can be approached through these methodological strategies:

• In vitro ubiquitination assays: Studies have successfully used immunopurified DPPA5 in combination with E1, E2-UbcH5a, and ubiquitin to investigate DPPA5 ubiquitination. This approach identified K35 as a critical lysine residue for polyubiquitination .

• Site-directed mutagenesis: To identify specific ubiquitination sites, researchers have created lysine-to-arginine mutations (K9R, K16R, K35R, K103R, K109R) in DPPA5 and assessed the impact on ubiquitination patterns. The K35R mutation specifically led to loss of ubiquitination in vitro .

• Trypsin-resistant ubiquitin binding entity (TR-TUBE) approaches: This technique has been used to protect polyubiquitin chains and confirm DPPA5 ubiquitination status. Using TR-TUBE plasmids tagged with HA in transfection experiments helps preserve and detect ubiquitination .

• Co-immunoprecipitation studies: This approach has successfully demonstrated the interaction between DPPA5 and the ubiquitin E3 ligase FBXO9. Using variants like FBXO9 ΔFbox and FBXO9 ΔTPR has helped elucidate the specific domains required for DPPA5 interaction .

• Functional assessment of modifications: Cellular reprogramming efficiency assays have shown that mutations affecting DPPA5 ubiquitination (e.g., K35R) attenuate reprogramming efficiency, providing functional validation of the significance of these modifications .

How can DPPA5 antibodies be incorporated into multi-parameter flow cytometry panels?

For integrating biotin-conjugated DPPA5 antibodies into multi-parameter flow cytometry panels, researchers should consider these methodological approaches:

• Panel design considerations:

  • Reserve the brightest fluorochromes (PE, APC) for markers with lower expression

  • Pair biotin-conjugated DPPA5 antibodies with streptavidin-conjugated fluorophores that have minimal spectral overlap with other markers in the panel

  • Include other pluripotency markers like OCT4, SSEA-3, SSEA-4, TRA-1-60, and GCTM-2

• Sample preparation protocol:

  • For extracellular markers: Label live cells for 30 minutes with primary antibodies including biotin-conjugated DPPA5

  • Wash in FACS buffer (PBS + 2% FBS)

  • Incubate with streptavidin-conjugated fluorophore for 30 minutes

  • Include 0.1% propidium iodide for viability assessment

• Compensation setup:

  • Use single-stained controls for each fluorophore, including the streptavidin-fluorophore used to detect biotin-conjugated DPPA5

  • Prepare an unstained control and FMO (fluorescence minus one) control for the DPPA5 channel

• Gating strategy:

  • First gate on viable cells (propidium iodide negative)

  • Apply forward/side scatter gates to eliminate debris and doublets

  • Analyze DPPA5 expression in conjunction with other pluripotency markers to identify subpopulations

• Controls for biotin staining:

  • Include a control with streptavidin-fluorophore only to account for endogenous biotin

  • Consider using avidin/biotin blocking kits if high background is observed

This approach has been validated in studies examining pluripotency markers in human embryonic stem cells and induced pluripotent stem cells .

What are common technical issues when using biotin-conjugated DPPA5 antibodies and how can they be addressed?

Researchers may encounter these challenges when working with biotin-conjugated DPPA5 antibodies:

• High background in streptavidin detection systems:

  • Cause: Endogenous biotin in samples or in serum-containing media

  • Solution: Implement avidin/biotin blocking steps before antibody incubation; reduce serum concentration during staining procedures; use biotin-free culture media for 24-48 hours before experiments

• Weak or absent signal in Western blotting:

  • Cause: Inadequate protein transfer, insufficient antibody concentration, or protein degradation

  • Solution: Verify transfer efficiency with Ponceau S staining; adjust antibody dilution within recommended range (1:500-1:2000); add protease inhibitors during sample preparation

• Multiple bands in Western blot:

  • Cause: Different modified forms of DPPA5, protein degradation, or non-specific binding

  • Solution: Use fresh samples with protease inhibitors; optimize blocking conditions; verify using a different DPPA5 antibody that recognizes a different epitope

• Cross-reactivity with other proteins:

  • Cause: Antibody binding to similar epitopes in related proteins

  • Solution: Validate specificity with knockout/knockdown controls; use immunoprecipitation to confirm specificity

• Inconsistent results between experiments:

  • Cause: Antibody degradation from improper storage or variable expression of DPPA5 in cell cultures

  • Solution: Aliquot antibodies upon receipt to avoid freeze-thaw cycles; standardize cell culture conditions; include positive controls in each experiment

What strategies can improve detection sensitivity when using biotin-conjugated DPPA5 antibodies?

To enhance detection sensitivity with biotin-conjugated DPPA5 antibodies, consider these evidence-based approaches:

• Signal amplification methods:

  • Use tyramide signal amplification (TSA) systems with streptavidin-HRP

  • Employ poly-HRP streptavidin conjugates instead of standard streptavidin

  • Consider biotin-streptavidin stacking methods for multilayer signal enhancement

• Sample preparation optimization:

  • Increase protein concentration in lysates for Western blotting

  • For immunofluorescence, optimize fixation methods (4% paraformaldehyde for 10-15 minutes is typically effective)

  • Implement antigen retrieval protocols if working with fixed tissues

• Incubation condition refinement:

  • Extend primary antibody incubation time to overnight at 4°C

  • Optimize blocking solutions to reduce background while preserving specific binding

  • Adjust detergent concentration in wash buffers to improve signal-to-noise ratio

• Detection system selection:

  • For fluorescence applications, select bright fluorophores like Alexa Fluor 488, 555, or 647

  • For enzymatic detection, consider supersensitive substrates like enhanced chemiluminescence (ECL) plus reagents

  • Use highly sensitive imaging systems with appropriate exposure settings

• Combined approaches:

  • For critical samples, consider using biotinylated secondary antibodies together with biotin-conjugated primary antibodies in sequential staining protocols

  • Implement computational methods for image enhancement and signal quantification

How can researchers effectively compare data obtained using different DPPA5 antibody formats?

When comparing results obtained with different DPPA5 antibody formats (biotin-conjugated, unconjugated, or tagged with other conjugates), researchers should implement these methodological approaches:

• Standardization of detection methods:

  • Use consistent cell types, culture conditions, and sample preparation protocols

  • Process samples in parallel when comparing different antibody formats

  • Standardize protein loading amounts for quantitative comparisons

• Calibration with standard samples:

  • Include a common positive control sample in all experiments

  • Generate standard curves using recombinant DPPA5 protein when conducting quantitative analyses

  • Use reference cell lines with known DPPA5 expression levels

• Epitope mapping considerations:

  • Account for differences in epitope recognition between antibodies (N-terminal vs. full-length recognition)

  • Document the specific amino acid sequences recognized by each antibody format

  • Consider how different epitopes might be affected by protein interactions or modifications

• Cross-validation strategies:

  • Confirm key findings using at least two different antibody formats

  • Validate with orthogonal methods (e.g., mRNA expression, reporter systems)

  • Document concordance and discrepancies between different antibody formats systematically

• Statistical approaches:

  • Implement appropriate normalization methods when comparing data from different antibody formats

  • Report variability metrics (standard deviation, coefficient of variation) for each antibody format

  • Use appropriate statistical tests to determine if differences between formats are significant

This methodical approach ensures reliable comparisons and helps identify format-specific artifacts that might influence experimental interpretations.

How are DPPA5 antibodies contributing to advances in cellular reprogramming research?

DPPA5 antibodies have become instrumental in advancing cellular reprogramming research through several key methodological applications:

• Monitoring reprogramming efficiency: DPPA5 antibodies have been used to track the emergence of pluripotent cells during reprogramming. Research has shown that DPPA5 expression correlates with successful reprogramming events, and mutations affecting its regulation (such as the K35R mutation that prevents ubiquitination) attenuate reprogramming efficiency .

• Identifying intermediate cell states: During the reprogramming process, DPPA5 antibodies help researchers identify and isolate cells transitioning between somatic and pluripotent states, allowing for molecular characterization of these intermediates.

• Evaluating culture conditions: DPPA5 antibodies have been utilized to assess various culture conditions for maintaining and inducing pluripotency, including comparing media like Naive Human Stem Cell Medium (NHSM), 5i/hLIF medium with FGF and Activin A (5i/L/FA), and RSeT medium .

• Understanding regulatory mechanisms: Research using DPPA5 antibodies has revealed regulatory mechanisms controlling pluripotency, such as the ubiquitin-proteasome system. The identification of FBXO9 as an E3 ligase that targets DPPA5 for ubiquitination at the K35 residue provides insight into the post-translational control of pluripotency factors .

• Correlation with other pluripotency markers: DPPA5 antibodies have helped establish the relationship between DPPA5 and other pluripotency factors, demonstrating high correlation with POU5F1 (OCT4) expression and surface markers like TRA-160 and SSEA-4 in human pluripotent stem cell cultures .

What are the emerging applications for DPPA5 antibodies in developmental biology research?

Emerging applications for DPPA5 antibodies in developmental biology research include:

• Single-cell analysis of early embryonic development: DPPA5 antibodies are being used in conjunction with single-cell transcriptomics to map the expression patterns during early embryonic development, providing insights into the temporal and spatial regulation of pluripotency genes .

• Investigation of naive versus primed pluripotency states: Research using DPPA5 antibodies has revealed distinct patterns of expression in naive versus primed human embryonic stem cells, contributing to our understanding of the molecular differences between these pluripotency states .

• Organoid development studies: DPPA5 antibodies are being implemented to track pluripotent cell populations during organoid formation, helping to understand the differentiation trajectories and maintenance of stem cell niches within these 3D structures.

• Cross-species comparative analyses: With DPPA5 antibodies showing reactivity across multiple species (human, mouse, rat, etc.), researchers are conducting comparative studies to understand evolutionary conservation of pluripotency mechanisms .

• Investigation of pseudogene function: DPPA5 pseudogenes located on chromosomes 10, 14, and 19 are being studied for potential functional roles, with DPPA5 antibodies helping to distinguish between authentic protein products and pseudogene-derived transcripts .