SNX24 Antibody

What is SNX24 and what experimental applications require its detection?

SNX24 is a sorting nexin family protein with a calculated molecular weight of 19-19.8 kDa that participates in cellular trafficking pathways. The protein contains 159 amino acids and is encoded by the SNX24 gene (Gene ID: 28966) . As a membrane-associated protein, SNX24 is commonly studied in vesicular transport, endosomal sorting, and protein trafficking investigations. Experimental applications requiring SNX24 antibodies typically include protein expression studies in normal and pathological states, subcellular localization, and interaction partner identification. The protein's relatively small size (observed molecular weight of 19 kDa) makes it important to select appropriately validated antibodies to ensure specific detection without cross-reactivity .

The reactivity profiles of commercially available SNX24 antibodies primarily cover human and mouse species, with predicted reactivity in other mammals. From the available technical data:

When selecting an antibody for cross-species applications, it is essential to verify experimental validation in the target species rather than relying solely on sequence homology predictions .

How should researchers validate SNX24 antibody specificity before experimental implementation?

Antibody validation is critical for ensuring experimental reproducibility and preventing misleading results. For SNX24 antibodies, a comprehensive validation approach should include:

• Knockout/Knockdown Controls: The gold standard for antibody validation is testing against samples where the target protein has been genetically eliminated or reduced. CRISPR-generated SNX24 knockout cell lines provide definitive negative controls to confirm antibody specificity .

• Western Blot Analysis: Verification that the antibody detects a band of the expected molecular weight (approximately 19 kDa for SNX24) in appropriate samples. Multiple cell lines should be tested, such as HEK-293, HeLa, MCF-7, and U2OS cells, which have been confirmed to express SNX24 .

• Multiple Antibody Comparison: When possible, compare results from different antibodies targeting distinct epitopes of SNX24 to confirm consistency in detection patterns .

• Immunoprecipitation-Mass Spectrometry: This approach can provide definitive evidence of antibody specificity by identifying pulled-down proteins.

The current antibody reproducibility crisis in biomedical research highlights that approximately 50% of commercial antibodies fail to meet basic standards for characterization, resulting in billions of dollars in research waste annually. Therefore, researchers must independently validate antibodies even when they come with manufacturer validation data .

What are the essential positive and negative controls for SNX24 antibody experiments?

Implementing appropriate controls is fundamental to generating reliable data with SNX24 antibodies:

Positive Controls:

• Cell lines with confirmed SNX24 expression (HEK-293, HeLa, MCF-7, U2OS)

• Mouse brain tissue (verified to express SNX24)

• Human ovary cancer tissue samples (for IHC applications)

Negative Controls:

• SNX24 knockout or knockdown cell lines/tissues (ideally CRISPR-generated)

• Primary antibody omission controls

• Isotype controls (using non-specific rabbit IgG for rabbit-derived SNX24 antibodies)

• Peptide competition assays using the immunizing peptide (particularly for antibodies generated against synthetic peptides, such as the Abbexa antibody produced against aa 14-43 from the N-terminal region)

The implementation of knockout controls has become increasingly accessible with CRISPR technologies, and researchers are strongly encouraged to incorporate these controls even when using previously validated antibodies .

What are the optimized protocols for SNX24 detection by Western blotting?

For optimal Western blot detection of SNX24, the following protocol parameters should be considered:

• Sample Preparation:

  • Lyse cells in RIPA or NP-40 buffer containing protease inhibitors

  • Include phosphatase inhibitors if post-translational modifications are being studied

  • Prepare tissues using mechanical homogenization followed by detergent solubilization

• Electrophoresis and Transfer:

  • Use 12-15% SDS-PAGE gels due to SNX24's relatively small size (19 kDa)

  • Transfer to PVDF or nitrocellulose membranes at 100V for 60-90 minutes or overnight at 30V

• Antibody Incubation:

  • Block with 5% non-fat milk or BSA in TBST for 1 hour at room temperature

  • Incubate with SNX24 primary antibody at dilutions:

    • Proteintech (16799-1-AP): 1:500-1:3000

    • Abbexa: 1:1000

  • Incubate overnight at 4°C with gentle rocking

  • Wash 3-5 times with TBST, 5 minutes each

  • Incubate with appropriate HRP-conjugated secondary antibody (typically anti-rabbit IgG)

  • Develop using ECL reagents and appropriate imaging system

• Expected Results:

  • SNX24 should be detected at approximately 19 kDa

  • Positive signal expected in HEK-293, HeLa, MCF-7, U2OS cells, and mouse brain tissue

For troubleshooting, titration of antibody concentration is recommended as the optimal dilution may vary depending on the specific experimental system and protein expression levels.

What are the recommended protocols for immunohistochemical detection of SNX24?

For immunohistochemical applications, the following optimized protocol is recommended:

• Tissue Preparation:

  • FFPE (formalin-fixed paraffin-embedded) tissue sections (4-6 μm thickness)

  • Deparaffinize and rehydrate through xylene and graded alcohols

• Antigen Retrieval:

  • Primary recommendation: TE buffer (pH 9.0)

  • Alternative method: citrate buffer (pH 6.0)

  • Heat-induced epitope retrieval (pressure cooker, microwave, or water bath)

• Staining Protocol:

  • Block endogenous peroxidase with 3% H₂O₂ in methanol (10 minutes)

  • Protein block with 5-10% normal serum (30 minutes)

  • Primary antibody incubation:

    • Proteintech (16799-1-AP): 1:50-1:500

    • Abbexa: 1:50-1:100

    • Incubate overnight at 4°C or 1-2 hours at room temperature

  • Detection using appropriate secondary antibody and detection system (ABC, polymer-based)

  • Develop with DAB or other chromogen

  • Counterstain with hematoxylin

• Validation:

  • Human ovary cancer tissue has been confirmed as positive control

  • Apply antibody dilutions at the lower end of the recommended range for initial optimization

  • Always include negative controls (primary antibody omission, non-specific IgG)

The Atlas Antibodies products indicate validation for IHC applications, suggesting these may also be suitable options, though specific dilution recommendations were not provided in the search results .

How can researchers address batch-to-batch variability when working with polyclonal SNX24 antibodies?

Batch-to-batch variability is a significant concern with polyclonal antibodies, including those targeting SNX24. This variability arises from the heterogeneous nature of the antibody population in different animals or bleeds, even when sold under the same catalog number. To address this challenge:

• Standardized Validation Protocol:

  • Establish a consistent validation protocol incorporating Western blot, IHC, and where possible, knockout controls

  • Document specific performance metrics (signal intensity, background, specificity) for each new batch

  • Maintain reference samples from previous successful experiments for direct comparison

• Lot Reservation:

  • When possible, reserve larger quantities of a single, well-performing lot for long-term studies

  • Document lot numbers used in all experiments to track potential sources of variability

• Multiple Antibody Approach:

  • Use two or more antibodies targeting different epitopes of SNX24 to confirm findings

  • Consider combining polyclonal antibodies with monoclonal options when available

• Transition Considerations:

  • When transitioning between batches, perform side-by-side comparisons using identical samples

  • Adjust protocols (dilutions, incubation times) as needed based on comparative performance

As noted in the eLife article, polyclonal antibodies present inherent limitations due to their "non-renewable nature and the complexity of the different antibodies present," which can significantly influence experimental reproducibility .

How can SNX24 knockout/knockdown models enhance experimental validity?

Knockout (KO) and knockdown (KD) models serve as essential tools for SNX24 antibody validation and functional studies:

• Antibody Validation Applications:

  • CRISPR-generated SNX24 KO cell lines provide definitive negative controls for antibody specificity

  • Partial knockdowns using siRNA or shRNA can determine antibody sensitivity thresholds

  • Isogenic cell line pairs (wild-type vs. KO) enable direct comparison under identical conditions

• Functional Study Applications:

  • SNX24 KO models allow investigation of phenotypic consequences of complete protein loss

  • Rescue experiments (reintroducing wild-type or mutant SNX24) can confirm specificity of observed phenotypes

  • KD models with varying degrees of depletion can reveal dose-dependent functions

• Implementation Strategies:

  • Generate stable KO cell lines using CRISPR-Cas9 targeting essential exons of SNX24

  • Verify knockout by genomic sequencing, mRNA analysis, and protein detection using validated antibodies

  • For in vivo studies, consider tissue-specific or inducible knockout systems to avoid developmental effects

How can researchers overcome common challenges in SNX24 immunodetection?

When working with SNX24 antibodies, several technical challenges may arise. The following strategies address common issues:

• Low Signal Intensity:

  • Increase antibody concentration (start with the higher end of recommended dilution range)

  • Extend primary antibody incubation time (overnight at 4°C)

  • Enhance signal amplification (HRP-polymer systems for IHC, high-sensitivity ECL for WB)

  • Optimize antigen retrieval conditions (test both recommended buffers: TE pH 9.0 and citrate pH 6.0)

  • Increase protein loading for Western blots (30-50 μg total protein)

• High Background/Non-specific Binding:

  • Increase blocking stringency (5% BSA instead of milk, longer blocking times)

  • Add 0.1-0.3% Triton X-100 to antibody dilution buffer to reduce hydrophobic interactions

  • Increase wash duration and frequency (5-6 washes, 10 minutes each)

  • Use antibody concentrations at the lower end of the recommended range

  • Consider antibody pre-adsorption with non-specific proteins

• Multiple Bands or Unexpected Molecular Weight:

  • Verify sample preparation (complete denaturation, fresh reducing agents)

  • Test different lysis buffers to ensure complete solubilization

  • Run gradient gels to improve resolution around the expected 19 kDa size

  • Compare with knockout controls to identify the specific SNX24 band

  • Consider post-translational modifications that may alter apparent molecular weight

• Tissue-Specific Detection Issues:

  • Optimize fixation time for tissues (overfixation can mask epitopes)

  • Test both recommended antigen retrieval methods (TE pH 9.0 and citrate pH 6.0)

  • Consider alternative antibodies targeting different epitopes of SNX24

  • Use positive control tissues with known expression (human ovary cancer tissue, mouse brain)

When implementing these strategies, it is advisable to change only one variable at a time and document all modifications to maintain experimental reproducibility.

How can researchers distinguish between SNX24 and related sorting nexin family members?

The sorting nexin family comprises multiple members with structural similarities that may lead to cross-reactivity issues. To ensure specific detection of SNX24:

• Epitope Selection Considerations:

  • Verify the immunogen used for antibody production (the Abbexa antibody targets aa 14-43 from the N-terminal region)

  • Select antibodies targeting unique regions of SNX24 with minimal homology to other sorting nexins

  • When possible, compare antibodies raised against different epitopes

• Cross-Reactivity Testing:

  • Test antibodies against recombinant proteins of closely related sorting nexins

  • Include cell lines with differential expression of sorting nexin family members

  • Consider using cell lines overexpressing individual sorting nexins as specificity controls

• Computational Analysis:

  • Perform sequence alignment of the immunizing peptide against the proteome to identify potential cross-reactive proteins

  • Use tools like BLAST to identify regions of homology between SNX24 and related proteins

  • Prioritize antibodies raised against regions with minimal sequence conservation

• Confirmatory Approaches:

  • Combine antibody-based detection with orthogonal methods (mass spectrometry, RNA expression)

  • Use knockout models of SNX24 and related sorting nexins to confirm specificity

  • Apply immunodepletion studies to verify antibody specificity

The careful selection and validation of antibodies against unique epitopes is particularly important for the sorting nexin family due to the presence of conserved functional domains across family members.