SBDS Antibody

What is SBDS protein and what are its primary cellular functions?

SBDS (Shwachman-Bodian-Diamond syndrome) protein is a highly conserved protein found across species from archaea to vertebrates and plants. It plays a crucial role in ribosome biogenesis and is essential for the assembly of mature ribosomes. Specifically, SBDS works together with EFL1 to trigger the GTP-dependent release of EIF6 from 60S pre-ribosomes in the cytoplasm . This process activates ribosomes for translation competence by allowing 80S ribosome assembly and facilitating EIF6 recycling to the nucleus.

Beyond its primary function in ribosome maturation, SBDS also contributes to:

• Maintenance of normal levels of protein synthesis

• Cellular stress resistance mechanisms

• Cellular response to DNA damage

• Cell proliferation regulation

Mutations in the SBDS gene are associated with Shwachman-Bodian-Diamond syndrome, an autosomal recessive disorder characterized by bone marrow dysfunction, exocrine pancreatic insufficiency, and a predisposition to myelodysplastic syndrome and acute myeloid leukemia.

What experimental applications are SBDS antibodies validated for?

SBDS antibodies have been validated for multiple experimental applications, with performance varying by specific antibody clone and manufacturer. Based on available commercial antibodies, the following applications are commonly supported:

Researchers should note that SBDS antibodies have been successfully tested with human, mouse, and rat samples, with human samples showing the highest consistency in experimental outcomes .

What is the molecular weight of SBDS protein and how does it appear on Western blots?

When performing Western blot analysis, SBDS protein typically appears as a distinct band. For example, using antibody ab154222, SBDS can be detected in various cell lysates including H1299 cells using 12% SDS-PAGE . Similarly, antibody 67200-1-Ig has been validated to detect SBDS in multiple cell lines including HeLa, HEK-293, HepG2, Jurkat, HSC-T6, NIH/3T3, and 4T1 cells .

For optimal results, researchers should include positive controls from well-characterized cell lines such as HeLa or HEK-293 where SBDS expression has been consistently documented.

How should SBDS antibodies be stored to maintain optimal activity?

Proper storage of SBDS antibodies is critical for maintaining their specificity and sensitivity. Based on manufacturer recommendations, the following storage conditions should be implemented:

How does SBDS deficiency affect cellular redox status and cell survival?

SBDS deficiency profoundly impacts cellular redox homeostasis, leading to dysregulation of reactive oxygen species (ROS) with significant consequences for cell growth and survival. Research using shRNA-mediated SBDS knockdown models has demonstrated that SBDS-deficient cells exhibit markedly increased baseline ROS levels compared to control cells .

This ROS dysregulation manifests in several interrelated cellular phenotypes:

• Enhanced susceptibility to apoptosis: SBDS-deficient cells show accelerated rates of both spontaneous and Fas-mediated apoptosis . Upon Fas stimulation, SBDS-knockdown cells experience a greater increase in ROS production compared to control cells, suggesting heightened sensitivity to death receptor signaling.

• Reduced cellular proliferation: Cells with SBDS deficiency demonstrate significantly slower growth rates, likely due to both increased cell death and altered cell cycle progression resulting from oxidative stress .

• Rescue by antioxidant treatment: Importantly, experimental administration of antioxidants can rescue SBDS-deficient cells from both increased apoptosis and reduced growth rates . This strongly implicates ROS dysregulation as a primary mechanism through which SBDS deficiency exerts its cellular effects.

The molecular pathway linking SBDS function to ROS regulation remains incompletely characterized, but may involve:

• Altered mitochondrial function secondary to disrupted protein synthesis

• Changes in translation of specific proteins involved in redox homeostasis

• Direct interactions between SBDS and components of cellular redox systems

These findings have significant implications for understanding the bone marrow failure and cancer predisposition observed in Shwachman-Diamond syndrome patients, as hematopoietic cells are particularly sensitive to oxidative stress .

What challenges exist in detecting SBDS gene variants using NGS, and how can they be overcome?

Detection of SBDS gene variants using next-generation sequencing (NGS) presents significant technical challenges due to the existence of a highly homologous pseudogene, SBDSP1, which shares 97% sequence identity with SBDS . This extensive homology creates specific complications in the NGS analytical pipeline:

• Mismapping of sequencing reads: Short NGS reads frequently align ambiguously between SBDS and SBDSP1, resulting in incorrect variant calling. This is particularly problematic for variants arising from gene conversion events .

• False negative variant calls: Pathogenic SBDS variants may be missed during conventional NGS analysis. For example, in a documented case (Case 3), two pathogenic SBDS variants (c.183_184delinsCT and c.201A>G) were correctly identified by Sanger sequencing but missed by standard NGS analysis because reads containing these variants were incorrectly aligned to SBDSP1 .

• False positive pseudogene variants: Simultaneously, normal sequence differences between SBDS and SBDSP1 may be mistakenly called as variants. In the same case, positions n.424 and n.533+10 of SBDSP1 were incorrectly called as heterozygous variants .

To overcome these challenges, researchers have developed specialized analytical approaches:

• Novel SBDS variant detection tools: Recently developed computational methods can properly analyze NGS data to correctly identify SBDS variants despite the homology challenges .

• Confirmation with orthogonal methods: Critical SBDS variants should be confirmed using Sanger sequencing or other targeted methods, particularly when clinical diagnosis is involved .

• Trio analysis: Analyzing patient samples alongside parental samples can help establish inheritance patterns of variants and distinguish true pathogenic changes from pseudogene artifacts .

• Custom bioinformatic pipelines: Specialized read mapping and variant calling algorithms that account for the high sequence similarity between SBDS and SBDSP1 can significantly improve detection accuracy .

These specialized approaches are essential when investigating Shwachman-Diamond syndrome through molecular methods, as conventional NGS analysis pipelines may miss clinically significant SBDS variants.

What is the role of SBDS in ribosome biogenesis and how does it affect translation?

SBDS plays a critical role in late-stage cytoplasmic maturation of 60S ribosomal subunits, functioning as a key factor in the final steps that prepare ribosomes for active translation. The molecular mechanisms through which SBDS facilitates this process have been elucidated through biochemical and structural studies:

• EIF6 release mechanism: SBDS functions in concert with elongation factor-like 1 (EFL1) to trigger the GTP-dependent release of eukaryotic initiation factor 6 (EIF6) from pre-60S ribosomal subunits in the cytoplasm . This release is essential because EIF6 prevents premature association of 40S and 60S ribosomal subunits.

• 80S ribosome assembly: Following EIF6 removal, the 60S subunit becomes competent to join with the 40S subunit, forming the complete 80S ribosome necessary for translation . Thus, SBDS directly enables the formation of translation-competent ribosomes.

• EIF6 recycling: Beyond simply removing EIF6, SBDS also facilitates its recycling back to the nucleus, where EIF6 is required for 60S rRNA processing and nuclear export . This creates a functional cycle that maintains appropriate ribosome production.

The consequences of SBDS dysfunction on translation include:

• Global reduction in protein synthesis rates: SBDS is "required for normal levels of protein synthesis" , with deficiency leading to broad translational defects.

• Potential translational bias: While not explicitly stated in the provided references, research in the field suggests SBDS deficiency may disproportionately affect the translation of specific mRNAs, particularly those involved in cellular stress response.

• Cellular stress sensitivity: The translational defects associated with SBDS deficiency likely contribute to the increased sensitivity to cellular stressors observed in SBDS-deficient cells .

These mechanisms help explain how SBDS mutations lead to the clinical manifestations of Shwachman-Diamond syndrome, as tissues with high protein synthesis requirements (such as bone marrow and pancreas) would be particularly vulnerable to SBDS dysfunction.

What are the optimal dilutions for various applications of SBDS antibodies?

Determining the optimal working dilution for SBDS antibodies is critical for achieving specific detection while minimizing background signals. Based on validated protocols, the following dilution ranges are recommended for different applications:

Important considerations for optimization include:

• Sample-dependent adjustment: The optimal dilution may vary depending on the sample type and SBDS expression level. For example, cells with high SBDS expression (like HeLa or HEK-293) may allow for higher antibody dilutions .

• Detection system influence: Enhanced chemiluminescence (ECL) systems with different sensitivities may require adjustment of antibody dilutions. More sensitive detection systems permit higher antibody dilutions.

• Titration recommendation: Manufacturers consistently recommend that "this reagent should be titrated in each testing system to obtain optimal results" . Initial experiments should include a dilution series to determine the optimal concentration for specific experimental conditions.

• Blocking conditions: Optimization of blocking conditions (typically 5% non-fat milk or BSA) in conjunction with antibody dilution can significantly improve signal-to-noise ratio.

How can researchers distinguish between SBDS and its pseudogene SBDSP1 in experimental settings?

Distinguishing between SBDS and its highly homologous pseudogene SBDSP1 presents significant challenges in both nucleic acid and protein-based experiments due to their 97% sequence identity . Researchers can employ several strategies to ensure specific detection of SBDS:

For DNA/RNA-based detection:

• Primer/probe design strategy: Design primers that target the seven base differences between SBDS and SBDSP1, focusing particularly on exonic regions where these differences occur .

• Long-read sequencing: When possible, utilize sequencing technologies that produce longer reads spanning multiple variant sites, allowing unambiguous assignment to either SBDS or SBDSP1.

• Custom bioinformatic pipelines: Implement specialized analytical approaches that account for the high sequence similarity, such as the novel SBDS variant detection tool mentioned in the research literature .

• Restriction enzyme analysis: Design strategies that exploit restriction enzyme recognition sites that differ between SBDS and SBDSP1 for validation studies.

For protein-based detection:

• Epitope selection: Choose antibodies raised against SBDS regions containing amino acid differences from SBDSP1 . Though not explicitly stated in the provided resources, manufacturers likely design immunogens that maximize specificity.

• Validation with knockdown controls: Verify antibody specificity using SBDS knockdown or knockout samples, which should show reduced or absent signal if the antibody is truly SBDS-specific .

• Expression pattern verification: SBDSP1 is a pseudogene and should not produce a functional protein. Therefore, detection of a protein at the expected molecular weight (29-31 kDa) in Western blot likely represents genuine SBDS .

• Multiple detection methods: Employ orthogonal techniques (e.g., mass spectrometry) alongside antibody-based detection to confirm protein identity based on peptide sequences unique to SBDS.

What controls should be included when conducting experiments with SBDS antibodies?

Robust experimental design with appropriate controls is essential for generating reliable data with SBDS antibodies. Researchers should implement the following controls based on the specific application:

For Western Blot applications:

• Positive controls: Include lysates from cell lines with well-documented SBDS expression such as HeLa, HEK-293, HepG2, or Jurkat cells . These samples establish the expected band size (29-31 kDa) and intensity.

• Negative controls: Implement SBDS knockdown or knockout samples using validated CRISPR/Cas9 systems or shRNA constructs . Several commercial options are available, including SBDS CRISPR/Cas9 KO Plasmids for both human and mouse models .

• Loading controls: Include detection of housekeeping proteins (β-actin, GAPDH, etc.) to normalize for total protein loading, particularly when comparing SBDS levels across different conditions.

• Molecular weight markers: Always run appropriate molecular weight standards to confirm the observed SBDS band matches the expected size of 29-31 kDa .

For Immunohistochemistry/Immunofluorescence:

• Tissue/cell specificity controls: Include tissues or cells known to express or lack SBDS. For instance, human breast cancer tissue has been validated for IHC applications with certain SBDS antibodies .

• Antibody specificity controls: Perform parallel staining omitting the primary antibody to assess potential background from the secondary detection system.

• Competing peptide controls: Where available, pre-incubate antibody with the immunizing peptide to demonstrate binding specificity.

• Subcellular localization validation: Confirm that observed SBDS staining patterns match the expected subcellular distribution, which includes both cytoplasmic and nuclear localization.

For functional studies:

• Genetic rescue controls: When studying SBDS-deficient models, include samples with re-expression of wild-type SBDS to demonstrate phenotype rescue .

• Dose-dependency: For treatments affecting SBDS function or expression, demonstrate dose-dependent effects to strengthen causality claims.

• Time-course analysis: Include temporal controls to distinguish immediate from delayed effects of SBDS manipulation.

• Antioxidant controls: When studying ROS-related phenotypes in SBDS-deficient systems, include antioxidant treatments to assess the contribution of ROS to observed phenotypes .

Implementation of these comprehensive controls enhances data reliability and facilitates proper interpretation of results in SBDS-focused research.

Why might Western blot detection of SBDS protein show unexpected results?

Western blot detection of SBDS protein may yield unexpected results due to various technical and biological factors. Understanding these potential issues can help researchers troubleshoot and correctly interpret their findings:

• Multiple or shifted bands: While SBDS typically appears at 29-31 kDa , researchers may observe:

  • Slightly shifted bands due to post-translational modifications

  • Higher molecular weight bands from protein complexes if sample denaturation is incomplete

  • Lower bands potentially representing degradation products or alternative splice variants

  • Non-specific bands from cross-reactivity with related proteins

• Weak or absent signal despite expected expression:

  • Inefficient protein extraction: SBDS participates in ribosome biogenesis and may be partially associated with nucleolar structures that require specialized extraction methods

  • Epitope masking: Post-translational modifications or protein-protein interactions may obscure antibody binding sites

  • Sample degradation: SBDS may be susceptible to proteolytic degradation during sample preparation

  • Suboptimal transfer conditions: High percentage gels or insufficient transfer time may impair transfer of SBDS to membranes

• Inconsistent results across cell types or tissues:

  • Varying expression levels: SBDS expression may differ substantially across cell types

  • Cell-type specific post-translational modifications affecting antibody recognition

  • Interference from sample-specific components that affect antibody binding

Troubleshooting recommendations:

• Optimize protein extraction: Use extraction buffers containing protease inhibitors and consider specialized nuclear extraction protocols for complete SBDS recovery.

• Validate with multiple antibodies: Test different SBDS antibodies targeting distinct epitopes to confirm findings .

• Include appropriate controls: Always run positive controls (e.g., HeLa or HEK-293 cell lysates) alongside experimental samples .

• Adjust blocking conditions: Test alternative blocking agents (milk vs. BSA) as certain antibodies may perform better with specific blocking protocols.

• Confirm specificity: Validate results with SBDS knockdown or knockout samples to ensure signals are specific .

How can researchers interpret SBDS expression changes in different experimental conditions?

• Baseline expression considerations:

  • SBDS is broadly expressed across tissues but may show cell type-specific regulation

  • Expression levels correlate with ribosomal biogenesis activity and cell proliferation status

  • Both transcript and protein levels should be assessed when possible, as post-transcriptional regulation may occur

• Interpreting decreased SBDS expression:

  • Partial reduction (50-80% of control): May represent physiological regulation or heterozygous mutation models

  • Severe reduction (>80%): Likely represents effective knockdown, knockout, or pathological conditions

  • Complete absence: Should be interpreted cautiously and verified with multiple detection methods, as residual expression is common even in knockdown models

• Evaluating increased SBDS expression:

  • Moderate increases may reflect enhanced ribosome biogenesis during cell growth or recovery

  • Substantial overexpression may indicate stress response, as SBDS has been implicated in cellular stress resistance

  • Consider whether increases are uniform across all cells or represent population shifts

• Contextualizing expression changes:

  • Correlate SBDS changes with functional outcomes such as ROS levels, apoptosis rates, and cell proliferation

  • Assess concurrently expressed ribosome biogenesis factors to determine whether changes are SBDS-specific or part of broader translational regulation

  • Determine reversibility of expression changes to distinguish adaptive from permanent alterations

• Technical validation approaches:

  • Confirm protein-level changes with multiple techniques (Western blot, immunofluorescence, flow cytometry)

  • Verify transcript changes using both qPCR and RNA-seq when possible

  • Use subcellular fractionation to determine whether changes affect specific cellular compartments

• Functional validation:

  • Rescue experiments: Test whether restoring SBDS expression reverses observed phenotypes

  • Dose-dependency: Establish whether biological effects correlate with the degree of SBDS expression change

  • Indirect effects: Consider whether SBDS changes might be secondary to alterations in cellular stress, growth rate, or other parameters

These comprehensive interpretation guidelines can help researchers accurately assess the significance of SBDS expression changes in their experimental systems and avoid common pitfalls in data analysis.

What approaches can resolve ambiguous SBDS gene variant detection in clinical and research settings?

Resolving ambiguous SBDS gene variant detection is crucial for both accurate diagnosis of Shwachman-Diamond syndrome and reliable research outcomes. The high homology between SBDS and its pseudogene SBDSP1 creates significant challenges that require specialized approaches:

• Advanced NGS analysis solutions:

  • Implement novel SBDS variant detection tools specifically designed to address the homology challenges in regular NGS data

  • Utilize longer read technologies that can span multiple differentiating positions between SBDS and SBDSP1

  • Apply custom bioinformatic pipelines with algorithms optimized for paralog-specific alignment

• Orthogonal validation methods:

  • Confirm NGS findings with Sanger sequencing using carefully designed primers that specifically amplify SBDS rather than SBDSP1

  • Employ digital droplet PCR with probes targeting SBDS-specific sequences

  • Consider RNA-based approaches to detect expressed variants when appropriate

• Family-based approaches:

  • Conduct trio analysis (patient plus parents) to establish inheritance patterns and phasing of variants

  • Examine segregation of variants with disease phenotype in extended families when available

  • Use parental samples to resolve compound heterozygosity vs. variants in cis

• Technical considerations for accurate detection:

  • Manual review of aligned reads in regions of high homology between SBDS and SBDSP1

  • Careful attention to coverage depth and variant allele frequencies, which may provide clues to misalignment issues

  • Cross-reference variant findings with known disease-causing mutations and their typical presentations

• Integrated diagnostic approach:

  • Combine genetic findings with clinical features, as certain phenotypes strongly suggest true SBDS pathogenic variants

  • Incorporate functional studies where possible, such as ribosome profiling or SBDS protein expression analysis

  • Consider the pattern of gene conversion events, as certain variant combinations frequently occur together (e.g., c.183_184delinsCT together with c.201A>G)

• Research and diagnostic standards:

  • Establish laboratory-specific validation protocols for SBDS variant detection

  • Maintain databases of confirmed variants with associated sequencing artifacts for reference

  • Regularly update bioinformatic pipelines as new tools for paralog discrimination become available

Implementation of these comprehensive approaches can significantly improve the accuracy of SBDS variant detection, reducing both false-negative and false-positive results that might otherwise occur due to the challenging genomic context of this clinically important gene .