SDR39U1 Antibody

What is SDR39U1 and what are its key structural characteristics?

SDR39U1 (Short chain dehydrogenase/reductase family 39U, member 1) is an epimerase family protein also known as C14orf124 or HCDI. It shares greater sequence similarity with the sugar epimerase family than with the short-chain dehydrogenases/reductases (SDR) family . The protein consists of 185 amino acids with a calculated molecular weight of approximately 35 kDa . SDR39U1 has three isoforms produced by alternative splicing with molecular weights of 35 kDa, 31 kDa, and 21 kDa . The protein sequence includes characteristic motifs associated with epimerase activity, as evident in its full amino acid sequence: MAYYQPSLTAEYDEDSPGGDFDFFSNLVTKWEAAARLPGDSTRQVVVRSGVVLGRGGGAMGHMLLPFRLGLGGPIGSGHQFFPWIHIGDLAGILTHALEANHVHGVLNGVAPSSATNAEFAQTLGAALGRRAFIPLPSAVVQAVFGRQRAIMLLEGQKVIPQRTLATGYQYSFPELGAALKEIVA .

What types of SDR39U1 antibodies are currently available for research applications?

Multiple SDR39U1 antibodies are available from various providers, targeting different epitopes and designed for specific applications. The table below summarizes key antibody types:

How should SDR39U1 antibodies be stored to maintain optimal activity?

For long-term stability and activity preservation of SDR39U1 antibodies, specific storage conditions must be followed. Most SDR39U1 antibodies should be stored at -20°C, where they typically remain stable for one year after shipment . Many are supplied in storage buffers containing PBS with 0.02% sodium azide and 50% glycerol at pH 7.3 to maintain stability . For antibodies supplied in smaller volumes (e.g., 20 µL), formulations may contain 0.1% BSA as an additional stabilizing agent .

It's important to note that aliquoting is generally unnecessary for -20°C storage of these antibodies, which simplifies handling procedures . For shipping purposes, wet ice is typically used . When retrieving antibodies from storage, avoid repeated freeze-thaw cycles as these can degrade antibody quality and reduce binding efficiency. Allow antibodies to equilibrate to room temperature before opening vials to prevent condensation that could introduce contamination or dilute the antibody solution.

What are the optimal dilution ratios for different applications of SDR39U1 antibodies?

Dilution ratios for SDR39U1 antibodies vary significantly based on the specific application, antibody type, and target tissue. Based on experimental validation data, the following application-specific dilutions are recommended:

These dilutions should be considered starting points, and researchers should optimize dilutions for their specific experimental systems . The optimization process should include positive and negative controls to determine signal-to-noise ratios. For challenging tissues or cell types, preliminary titration experiments with serial dilutions ranging from 1:100 to 1:2000 can help identify optimal concentrations that maximize specific signal while minimizing background.

How can I validate the specificity of SDR39U1 antibodies for my experimental system?

Validating antibody specificity is crucial for reliable results. For SDR39U1 antibodies, implement multiple complementary validation strategies:

• Western blot analysis: Look for a predominant band at 35 kDa (main isoform), with possible additional bands at 31 kDa and 21 kDa representing alternative splice variants . Validation should include:

  • Positive control tissues known to express SDR39U1 (e.g., human adrenal gland)

  • Negative control tissues with minimal expression

  • Blocking peptide competition to confirm specificity

• RNA interference: Transfect cells with SDR39U1-specific siRNA and verify reduced signal with the antibody compared to non-targeting siRNA controls. This approach confirms that the antibody detects the intended target.

• Recombinant expression: Overexpress tagged SDR39U1 in cell lines with low endogenous expression and verify co-localization of antibody signal with the tag-specific antibody.

• Immunoprecipitation followed by mass spectrometry: This confirms that the antibody captures the intended protein rather than cross-reactive species.

• Multiple antibody validation: Use different antibodies targeting distinct epitopes of SDR39U1 (e.g., N-terminal, C-terminal, and middle region antibodies) and verify consistent results across methods.

When documenting validation results, include complete methodology, positive and negative controls, and quantitative assessments of specificity to ensure reproducibility.

What protocol modifications are necessary when detecting different SDR39U1 isoforms?

SDR39U1 exists in three isoforms (35 kDa, 31 kDa, and 21 kDa) produced by alternative splicing , requiring specific protocol adjustments for effective detection:

• Antibody selection: Choose antibodies that target conserved regions present in all isoforms for pan-isoform detection, or epitope-specific antibodies for selective isoform identification. For example:

  • X-Q86TZ5-N targets the N-terminus and may detect all isoforms if this region is conserved

  • X-Q86TZ5-C targets the C-terminus and may miss truncated isoforms

• Gel resolution optimization: For Western blotting, use 12-15% polyacrylamide gels for better separation of closely sized isoforms (35 kDa vs. 31 kDa).

• Extended running time: Increase electrophoresis duration by 20-30% to enhance separation between the 31 kDa and 35 kDa bands.

• Two-dimensional electrophoresis: For complex samples, consider 2D-PAGE to separate isoforms that differ in isoelectric point as well as molecular weight.

• RT-PCR verification: Design primers that can distinguish between splice variants and perform RT-PCR alongside protein detection to correlate RNA and protein expression patterns.

• Enrichment strategies: For low-abundance isoforms, consider immunoprecipitation with isoform-specific antibodies before Western blotting to increase detection sensitivity.

When reporting results, clearly document which isoforms were detected and include molecular weight markers to facilitate interpretation.

What are the common causes of high background when using SDR39U1 antibodies for immunohistochemistry, and how can they be resolved?

High background in immunohistochemistry with SDR39U1 antibodies can arise from various sources. The following table outlines common causes and their solutions:

For particularly challenging samples, consider modifying antigen retrieval methods. For SDR39U1 detection, compare heat-induced epitope retrieval using citrate buffer (pH 6.0) versus EDTA buffer (pH 9.0) to determine optimal conditions. Additionally, reducing primary antibody incubation temperature from room temperature to 4°C while extending incubation time can improve signal-to-noise ratio.

Why might Western blot detection of SDR39U1 show unexpected band patterns, and how should these results be interpreted?

Western blot analysis of SDR39U1 may reveal complex band patterns beyond the expected 35 kDa, 31 kDa, and 21 kDa isoforms . These can result from various biological and technical factors:

• Post-translational modifications: Higher molecular weight bands may indicate phosphorylation, glycosylation, or ubiquitination. Verify with:

  • Phosphatase treatment (for phosphorylation)

  • Glycosidase treatment (for glycosylation)

  • Immunoprecipitation followed by modification-specific antibodies

• Protein complexes: If sample preparation is not fully denaturing, SDR39U1 may remain in protein complexes. To resolve:

  • Increase SDS concentration to 2% in sample buffer

  • Extend boiling time to 10 minutes

  • Add reducing agents like DTT (10 mM)

• Proteolytic degradation: Lower-than-expected molecular weight bands may indicate sample degradation. Prevent by:

  • Adding fresh protease inhibitors during extraction

  • Maintaining samples at 4°C throughout preparation

  • Avoiding repeated freeze-thaw cycles

• Cross-reactivity: Bands at unexpected molecular weights may represent cross-reactivity with related proteins. Address by:

  • Performing peptide competition assays

  • Using alternative antibodies targeting different epitopes

  • Confirming results with knockout/knockdown controls

• Isoform-specific expression: Different tissues may express varying ratios of the three known isoforms. Document tissue-specific expression patterns systematically across multiple samples.

When unexpected band patterns persist despite troubleshooting, consider mass spectrometry analysis of the corresponding gel regions to identify the proteins definitively.

How can SDR39U1 antibodies be optimized for multiplex immunofluorescence to study protein interactions in situ?

Multiplex immunofluorescence with SDR39U1 antibodies requires careful optimization to maintain specificity while enabling co-detection of interaction partners. Implement the following strategies:

• Antibody compatibility assessment:

  • Test SDR39U1 antibodies from different host species (e.g., rabbit polyclonal HPA044294 and mouse monoclonal X-Q86TZ5 variants )

  • Evaluate different clones for epitope compatibility and similar staining conditions

  • Perform single-staining controls before multiplexing

• Sequential immunostaining protocol:

  • Begin with the lowest concentration antibody (typically SDR39U1 antibodies at 0.25-2 μg/mL for IF )

  • Apply tyramide signal amplification (TSA) if signal strength varies significantly between targets

  • Include a heat or chemical inactivation step between rounds of staining to prevent cross-reactivity

• Fluorophore selection for optimal spectral separation:

  • Use fluorophores with minimal spectral overlap (e.g., FITC for SDR39U1, Cy3 for first interaction partner, Cy5 for second interaction partner)

  • Include single-color controls to establish spectral unmixing parameters

  • Consider using quantum dots for narrow emission spectra if available

• Image acquisition optimization:

  • Apply sequential scanning rather than simultaneous acquisition

  • Set exposure times individually for each channel to balance signal intensities

  • Use appropriate negative controls to set threshold values

• Colocalization analysis:

  • Calculate Pearson's or Mander's coefficients to quantify spatial overlap

  • Implement proximity ligation assay (PLA) to verify direct protein interactions within 40 nm

  • Use deconvolution algorithms to improve spatial resolution before colocalization analysis

These approaches can reveal SDR39U1's subcellular localization patterns and potential interaction partners in different tissue contexts, providing insights into its functional roles.

What considerations should be taken when using SDR39U1 antibodies for chromatin immunoprecipitation (ChIP) studies?

Although SDR39U1 is not a known transcription factor, investigating its potential chromatin associations requires special considerations when adapting antibodies for ChIP applications:

• Antibody validation for ChIP compatibility:

  • Verify nuclear localization of SDR39U1 using immunofluorescence with antibodies like HPA044294

  • Perform preliminary IP experiments to confirm antibody can capture native protein

  • Test antibody functionality in the presence of formaldehyde-fixed chromatin

• Crosslinking optimization:

  • Start with standard 1% formaldehyde for 10 minutes

  • Consider dual crosslinking with additional DSG (disuccinimidyl glutarate) for more transient interactions

  • Test multiple chromatin fragmentation methods (sonication vs. enzymatic digestion)

• Protocol adaptation:

  • Increase antibody amounts (5-10 μg per reaction) compared to standard IP

  • Extend incubation times (overnight at 4°C) to improve capture efficiency

  • Include additives like BSA (0.5%) and non-ionic detergents to reduce background

• Stringent controls:

  • Use IgG from the same species as the SDR39U1 antibody for negative controls

  • Include input controls at multiple concentrations

  • Perform SDR39U1 knockdown controls to confirm specificity of enrichment

• Downstream analysis considerations:

  • Start with qPCR analysis of regions of interest before proceeding to sequencing

  • For ChIP-seq, prepare biological replicates and perform correlation analysis

  • Consider spike-in normalization for quantitative comparisons

If SDR39U1 is found to associate with chromatin, these studies could reveal unexpected regulatory functions beyond its known enzymatic role in carbohydrate metabolism, potentially expanding understanding of its cellular functions.

How can SDR39U1 antibodies be utilized in immunoprecipitation-mass spectrometry (IP-MS) to identify novel protein interactions?

IP-MS with SDR39U1 antibodies can uncover previously unknown protein interactions by following these methodological guidelines:

• Antibody selection criteria:

  • Choose antibodies with proven IP efficiency

  • Select antibodies targeting different epitopes (N-terminal, middle region, C-terminal) to cross-validate interactions

  • Verify antibody specificity by Western blot before IP-MS

• Lysis buffer optimization:

  • Test multiple lysis conditions:

    • Stringent: RIPA buffer (high detergent, disrupts weaker interactions)

    • Moderate: NP-40 buffer (preserves most protein complexes)

    • Mild: Digitonin buffer (maintains membrane-associated complexes)

  • Include phosphatase inhibitors to preserve modification-dependent interactions

  • Consider crosslinking agents for transient interactions

• IP protocol refinements:

  • Pre-clear lysates with irrelevant antibody and protein A/G beads

  • Determine optimal antibody-to-lysate ratios (starting with 5 μg antibody per mg protein)

  • Include appropriate controls:

    • IgG control from same species as SDR39U1 antibody

    • Cell lines with SDR39U1 knockdown/knockout

    • Competition with immunizing peptide

• MS sample preparation:

  • Perform on-bead digestion to minimize contamination

  • Include brief detergent wash steps to reduce non-specific binding

  • Consider SILAC or TMT labeling for quantitative comparison between conditions

• Data analysis strategy:

  • Filter against IgG control interactome to remove non-specific binders

  • Apply statistical thresholds (fold change >2, p<0.05) to identify significant interactions

  • Validate top hits by reciprocal IP and co-immunofluorescence

  • Perform functional classification and network analysis of interactors

This approach can reveal SDR39U1's position within cellular protein networks and provide clues about its functional roles beyond current annotations in epimerase activity and sugar metabolism.

How do different SDR39U1 antibody types compare in sensitivity and specificity across various applications?

Different antibody types offer distinct advantages for SDR39U1 detection depending on the application. The table below compares performance characteristics across key methods:

When selecting between these options, consider:

The ideal approach often involves using multiple antibody types in parallel to leverage their complementary strengths and confirm findings through independent methods.

What are the advantages and limitations of using SDR39U1 antibodies compared to alternative detection methods?

SDR39U1 detection can be accomplished through various methodologies, each with distinct strengths and limitations:

For comprehensive SDR39U1 characterization, an integrated approach is recommended:

• Use antibody-based methods for initial localization and expression studies

• Confirm key findings with orthogonal techniques (mRNA analysis, MS)

• For functional studies, combine antibody detection with genetic approaches (knockdown/knockout)

• For novel interaction studies, validate antibody-based findings with tag-based reciprocal experiments

What emerging technologies may enhance the utility of SDR39U1 antibodies for single-cell and spatial analysis?

Recent technological advances are expanding applications for SDR39U1 antibodies beyond traditional methods:

• Mass cytometry (CyTOF) integration:

  • Conjugate SDR39U1 antibodies with rare earth metals

  • Enables simultaneous detection of 40+ proteins in single cells

  • Allows correlation of SDR39U1 expression with comprehensive cell phenotyping

  • Implementation steps: Validate metal-conjugated antibodies against standard IF, establish optimal staining parameters, develop antibody panels including lineage markers

• Spatial transcriptomics-antibody hybridization:

  • Combine SDR39U1 antibody detection with in situ RNA localization

  • Correlates protein expression with transcriptome at near-cellular resolution

  • Technologies to implement: 10x Visium with immunofluorescence, Slide-seq with antibody detection

  • Validation approach: Compare spatial patterns to conventional IHC, establish RNA-protein correlation maps

• Super-resolution microscopy optimization:

  • Adapt SDR39U1 antibodies for STORM/PALM techniques

  • Achieves 10-20 nm resolution of protein localization

  • Reveals previously undetectable subcellular distribution patterns

  • Protocol modifications: Use bright, photoswitchable fluorophores, optimize antibody density, employ drift correction

• Microfluidic antibody analysis:

  • Integrate SDR39U1 antibodies into microfluidic platforms

  • Enables high-throughput single-cell protein quantification

  • Reduces antibody consumption while increasing analytical throughput

  • Implementation strategy: Validate antibody performance in microfluidic channels, establish detection limits, develop multiplexed protocols

• Antibody-based proximity labeling:

  • Convert SDR39U1 antibodies to proximity labeling tools (APEX2, BioID)

  • Maps protein interaction networks in native cellular environments

  • Identifies transient or weakly-associated partners

  • Development pathway: Generate antibody-enzyme fusions, validate labeling efficiency, optimize reaction conditions

These emerging technologies will enable researchers to study SDR39U1 with unprecedented spatial and molecular resolution, potentially revealing new aspects of its function in cellular physiology and disease contexts.

What are the methodological considerations for using SDR39U1 antibodies in cross-species comparative studies?

Cross-species research with SDR39U1 antibodies requires careful methodological planning due to sequence variations that can affect epitope recognition:

• Epitope conservation analysis:

  • Align SDR39U1 sequences across target species using multiple sequence alignment tools

  • Calculate percent identity within antibody epitope regions

  • For HPA044294, analyze conservation within the immunogen sequence (RLPGDSTRQVVVRSGVVLGRGGG...)

  • Predict potential cross-reactivity based on epitope conservation percentages (>80% identity suggesting likely cross-reactivity)

• Antibody selection strategy:

  • Prioritize antibodies validated across multiple species (e.g., Proteintech 16585-1-AP shows reactivity with human, mouse, and rat samples)

  • Target highly conserved protein domains for maximum cross-species utility

  • Consider generating new antibodies against universally conserved epitopes for novel species studies

• Validation requirements for each species:

  • Perform Western blot analysis in each target species with positive and negative control tissues

  • Implement knockdown/knockout controls in available model systems

  • Compare observed molecular weights with species-specific predictions based on cDNA sequences

  • Document species-specific band patterns that may reflect different post-translational modifications

• Protocol optimization across species:

  • Adjust antibody concentrations independently for each species (typically higher for less conserved epitopes)

  • Modify antigen retrieval conditions based on species-specific tissue fixation responses

  • Evaluate multiple blocking reagents to minimize species-specific background

  • Test incubation times and temperatures to optimize signal-to-noise ratio

• Quantitative comparison considerations:

  • Establish species-specific standard curves using recombinant proteins when possible

  • Implement normalization strategies based on conserved housekeeping proteins

  • Account for differences in antibody affinity when comparing expression levels across species

  • Report results as relative changes within species rather than absolute comparisons

These methodological considerations ensure that cross-species comparisons of SDR39U1 expression and function yield valid biological insights rather than artifacts of antibody performance variation.