SMIM29 Antibody, Biotin conjugated

What is SMIM29 protein and what are its key characteristics?

SMIM29 (Small Integral Membrane Protein 29) is a membrane-localized protein with a canonical length of 102 amino acid residues and a molecular weight of approximately 11.6 kDa in humans. Also known as uncharacterized protein C6orf1, SMIM29 has up to two different isoforms reported in the literature . The protein exhibits tissue-specific expression patterns, being found predominantly in spleen, thymus, prostate, testis, uterus, small intestine, colon, and peripheral blood leukocytes . SMIM29 undergoes post-translational modifications, particularly glycosylation, which may affect its function and detection . Evolutionary conservation is evidenced by the presence of orthologs in multiple species including mouse, rat, chimpanzee, and chicken, suggesting important biological functions across vertebrates .

What are the primary applications for SMIM29 Antibody, Biotin conjugated?

SMIM29 Antibody, Biotin conjugated is primarily utilized in enzyme-linked immunosorbent assays (ELISA) for the detection and quantification of SMIM29 protein . The biotin conjugation enables high-sensitivity detection through streptavidin or avidin-based detection systems. While ELISA represents the validated application for the biotin-conjugated format, non-conjugated SMIM29 antibodies have broader application profiles including Western Blotting, Immunofluorescence, and Immunohistochemistry . Researchers should consider that while the biotinylated format provides advantages in detection sensitivity, it may present challenges in contexts with high endogenous biotin, requiring careful experimental design and appropriate controls .

What is the optimal storage and handling protocol for SMIM29 Antibody, Biotin conjugated?

For maximum stability and activity retention, SMIM29 Antibody, Biotin conjugated should be stored at -20°C or -80°C immediately upon receipt . The antibody is supplied in liquid form containing 50% glycerol, 0.01M PBS (pH 7.4), and 0.03% Proclin 300 as a preservative . To minimize freeze-thaw cycles that can compromise antibody integrity, aliquoting the antibody into smaller volumes prior to freezing is recommended. When handling the antibody for experiments, thaw aliquots on ice and keep cold during use. For short-term storage (1-2 weeks), the antibody can be kept at 4°C, but extended storage at this temperature is not recommended due to potential biotin degradation. Always centrifuge briefly before opening the vial to ensure solution homogeneity and prevent sample loss.

How should researchers control for endogenous biotin interference when using SMIM29 Antibody, Biotin conjugated?

Endogenous biotin interference presents a significant challenge when using biotinylated antibodies like SMIM29 Antibody, Biotin conjugated, particularly in mitochondria-rich samples where biotinylated enzymes are abundant . To control for this interference, researchers should implement a multi-faceted approach:

This systematic approach not only controls for potential false positives but also provides internal validation of SMIM29 detection specificity . When analyzing tissues particularly rich in mitochondria (heart, liver, kidney), additional blocking steps may be necessary to achieve optimal signal-to-noise ratios.

What optimized protocol should be followed for ELISA using SMIM29 Antibody, Biotin conjugated?

Based on technical specifications and standard ELISA protocols using biotinylated antibodies, the following optimized procedure is recommended:

• Plate Coating: Coat 96-well plate with capture antibody (anti-SMIM29) at 1-10 μg/mL in carbonate/bicarbonate buffer (pH 9.6) overnight at 4°C.

• Blocking: Block with 1-5% BSA or 5% non-fat dry milk in PBS-T (PBS + 0.05% Tween-20) for 1-2 hours at room temperature.

• Sample Preparation: Prepare recombinant SMIM29 standards (0-1000 ng/mL) and dilute unknown samples appropriately in sample diluent (typically PBS-T with 1% BSA).

• Sample Incubation: Add standards and samples, incubate for 2 hours at room temperature or overnight at 4°C.

• Detection Antibody: Add SMIM29 Antibody, Biotin conjugated diluted to the optimized concentration (typically 1-2 μg/mL) in antibody diluent (PBS-T with 1% BSA), incubate for 1-2 hours at room temperature .

• Signal Development:

  • Add streptavidin-HRP (1:1000 to 1:5000 dilution) for 30-60 minutes

  • Wash extensively to reduce background

  • Add substrate solution (TMB)

  • Stop reaction with stop solution (2N H₂SO₄)

  • Read absorbance at 450 nm with 570 nm reference wavelength

• Critical Considerations:

  • Include a biotin blocking step if using samples with high endogenous biotin

  • Ensure adequate washing (5-7 washes) after each step to minimize background

  • Optimize antibody concentration for each new lot

This protocol can be adjusted based on specific sample types and detection sensitivity requirements.

How can SMIM29 Antibody, Biotin conjugated be validated for specificity in experimental systems?

Comprehensive validation of SMIM29 Antibody, Biotin conjugated specificity requires multiple complementary approaches:

• Western Blot Analysis: Though not the primary application for the biotinylated format, western blot using the non-biotinylated variant can confirm antibody recognition of a single band at the expected molecular weight (11.6 kDa for SMIM29) . This provides crucial information about specificity before proceeding with the biotinylated format.

• Peptide Competition Assay: Pre-incubation of the antibody with excess recombinant SMIM29 protein (specifically amino acids 42-102, which represent the immunogen region) should abolish specific signal in the intended application .

• Knockout/Knockdown Controls: Testing the antibody in SMIM29 knockout or knockdown systems provides the most stringent validation. Significant signal reduction should be observed compared to wild-type samples.

• Tissue Panel Analysis: Testing the antibody across tissues with known differential expression of SMIM29 (high in spleen, thymus, prostate, testis, uterus, small intestine, colon, and peripheral blood leukocytes; low in other tissues) should yield corresponding signal patterns .

• Cross-Species Reactivity: Confirming reactivity with predicted orthologs in mouse, rat, chimpanzee, or chicken can provide additional validation when appropriate controls are included .

• Parallel Antibody Comparison: Comparing results with alternative SMIM29 antibodies from different sources or those recognizing different epitopes can provide convergent validation.

Implementation of these validation strategies ensures experimental results reflect true SMIM29 biology rather than artifacts or non-specific binding.

How can SMIM29 Antibody, Biotin conjugated be utilized in cell sorting applications?

SMIM29 Antibody, Biotin conjugated can be effectively incorporated into Buoyancy-Activated Cell Sorting (BACS) protocols by adapting methodologies used for other biotinylated antibodies . This approach utilizes the strong biotin-avidin interaction to couple antibodies to microbubbles for cell separation.

The process involves:

• Microbubble Preparation: Prepare biotinylated albumin microbubbles (biotin-MBs) through sonication of a solution containing biotinylated human serum albumin and appropriate gas (e.g., C₃F₈) .

• Antibody Conjugation System:

  • Incubate biotin-MBs with avidin (2 mg/mL) overnight at 4°C

  • Subsequently incubate with SMIM29 Antibody, Biotin conjugated for at least 2 hours at 4°C

• Cell Targeting: Mix the SMIM29-targeted biotin-MBs with the cell suspension containing SMIM29-expressing cells for 15 minutes at room temperature .

• Separation: Allow the microbubble-bound cells to float to the surface based on buoyancy, while unbound cells remain in solution.

• Collection and Analysis: Carefully collect the floating fraction (SMIM29-positive) and the non-floating fraction (SMIM29-negative) for further analysis or culture.

What are the optimal conditions for using SMIM29 Antibody, Biotin conjugated in multiplex immunoassays?

When incorporating SMIM29 Antibody, Biotin conjugated into multiplex immunoassays, careful optimization of several parameters is essential to achieve specific detection without cross-reactivity:

• Detection System Selection: For multiplexing, avoid using multiple biotin-conjugated antibodies simultaneously unless employing sequential detection strategies. Alternative detection chemistries (e.g., direct fluorophore conjugates) should be used for other targets in the multiplex panel.

• Signal Separation Strategies:

• Biotin Blocking: When multiplexing with other detection systems in biotin-rich samples, include an avidin/streptavidin blocking step followed by a biotin blocking step before introducing any biotinylated antibodies .

• Cross-Reactivity Testing: Perform single-antibody controls alongside multiplex experiments to identify any potential cross-reactivity or interference between detection systems.

• Signal Amplification Balancing: Adjust streptavidin-conjugate concentration and incubation time to achieve comparable signal intensity with other detection methods in the multiplex panel.

These optimizations ensure accurate simultaneous detection of SMIM29 alongside other targets of interest in complex biological samples.

How can researchers troubleshoot weak or inconsistent signals when using SMIM29 Antibody, Biotin conjugated?

When encountering weak or inconsistent signals with SMIM29 Antibody, Biotin conjugated, researchers should systematically address potential issues using this troubleshooting guide:

• Antibody Functionality Assessment:

  • Verify antibody integrity through dot blot analysis with recombinant SMIM29 protein

  • Check for biotin conjugation efficiency using streptavidin-based detection

  • Test antibody using positive control samples with known SMIM29 expression

• Protocol Optimization Parameters:

• Signal Amplification Strategies:

  • Implement tyramide signal amplification (TSA) with biotinylated tyramine

  • Use poly-HRP streptavidin rather than standard streptavidin-HRP

  • Consider biotin-streptavidin layering (biotin-streptavidin-biotin) for additional amplification

• Epitope Retrieval Enhancement:

  • For fixed tissues/cells, compare different antigen retrieval methods (heat-induced vs. enzymatic)

  • Test different pH conditions for retrieval buffers (citrate pH 6.0 vs. EDTA pH 9.0)

  • Extend retrieval time in challenging samples

• Endogenous Biotin Management:

  • Implement avidin/biotin blocking kit before antibody application

  • Use streptavidin mutants with reduced binding to endogenous biotin

Systematic application of these troubleshooting strategies should identify and resolve the specific factors limiting SMIM29 detection in experimental systems.

How does SMIM29 Antibody, Biotin conjugated compare with non-conjugated formats for different applications?

A comparative analysis of SMIM29 Antibody in biotinylated versus non-conjugated formats reveals application-specific advantages and limitations:

The selection between formats should be guided by the specific experimental requirements, tissue context, and desired sensitivity/specificity balance. For novel applications or challenging samples, side-by-side comparison of both formats is recommended to determine the optimal approach.

What is the current understanding of SMIM29 function and how does antibody detection contribute to this research?

While SMIM29 remains relatively uncharacterized functionally (hence its alternative name "uncharacterized protein C6orf1"), antibody-based detection methods provide crucial insights into its biology:

• Expression Pattern Analysis: SMIM29 Antibody detection has revealed tissue-specific expression in spleen, thymus, prostate, testis, uterus, small intestine, colon, and peripheral blood leukocytes, suggesting potential roles in immune function, reproduction, and epithelial biology .

• Subcellular Localization: Antibody-based immunofluorescence studies confirm SMIM29's membrane localization, providing clues to its functional context .

• Post-Translational Modifications: Detection of glycosylation patterns through antibody-based methods points to potential regulatory mechanisms and protein-protein interactions .

• Evolutionary Conservation: Cross-reactivity testing with orthologs in mouse, rat, chimpanzee, and chicken indicates evolutionarily conserved functions worthy of investigation .

• Disease Associations: Antibody-based protein detection complements genomic studies in identifying potential disease associations, though specific SMIM29-disease links remain to be fully characterized.

Future research directions facilitated by specific antibody detection include:

• Interactome mapping via co-immunoprecipitation

• Expression correlation with disease progression

• Functional studies through antibody-mediated inhibition

• Therapeutic targeting potential assessment

The development of well-validated antibody tools, including the biotin-conjugated format, is essential for advancing our understanding of this understudied protein.

What methodological considerations apply when quantifying SMIM29 expression levels using antibody-based approaches?

Accurate quantification of SMIM29 expression requires careful consideration of multiple methodological factors:

• Quantification Method Selection:

• Standard Curve Development: For absolute quantification via ELISA, recombinant SMIM29 protein (aa 42-102) should be used to generate standard curves, ensuring the same epitope recognition as test samples .

• Normalization Strategies:

  • Cell number/density normalization via DNA content or nuclei counting

  • Total protein normalization with consistent extraction efficiency

  • Housekeeping protein normalization with verified stable expression

  • Multiple reference gene/protein approach for improved accuracy

• Technical Replication Requirements:

  • Minimum triplicate technical replicates recommended

  • Inter-assay calibrators included across experimental batches

  • Standard curve on each experimental plate/membrane

• Statistical Analysis Considerations:

  • Log-transformation of concentration data often improves normality

  • Non-parametric statistics recommended for small sample sizes

  • Analysis of lower detection limits and censored data

How might SMIM29 Antibody, Biotin conjugated be utilized in emerging research technologies?

SMIM29 Antibody, Biotin conjugated has potential applications in several cutting-edge research technologies:

• Spatial Transcriptomics Integration: The biotin conjugate can serve as a bridge between protein detection and spatial transcriptomics through sequential immunofluorescence and in situ sequencing protocols, allowing correlation between SMIM29 protein expression and local transcriptional landscapes.

• Single-Cell Proteomics: Biotinylated antibodies can be incorporated into microfluidic-based single-cell proteomics workflows, enabling analysis of SMIM29 expression heterogeneity at the individual cell level in complex tissues.

• Proximity Labeling Approaches: The biotin tag can be utilized for proximity-dependent biotinylation when coupled with promiscuous biotin ligases (BioID or TurboID), facilitating identification of SMIM29 interaction partners in living cells.

• Targeted Protein Degradation: Biotinylated antibodies can be coupled with streptavidin-proteasome adaptors in targeted protein degradation systems, enabling selective depletion of SMIM29 for functional studies.

• Nanobody Development: The well-characterized epitope recognition provided by validated biotinylated antibodies can guide the development of nanobodies against SMIM29, offering improved penetration for in vivo imaging.