SECISBP2 Antibody

What is SECISBP2 and what is its role in selenoprotein synthesis?

SECISBP2 (Selenocysteine insertion sequence-binding protein 2) is an mRNA-binding protein that facilitates the incorporation of selenocysteine into proteins. It functions by binding to the SECIS (selenocysteine insertion sequence) element present in the 3'-UTR of mRNAs encoding selenoproteins . The selenocysteine incorporation mechanism involves several steps: (1) SECISBP2 binds the SECIS sequence when the 80S ribosome encounters an in-frame UGA codon, (2) SECISBP2 contacts the RPS27A/eS31 of the 40S ribosome before ribosome stalling, (3) GTP-bound EEFSEC delivers selenocysteinyl-tRNA(Sec) to the 80S ribosome, and (4) after GTP hydrolysis, selenocysteinyl-tRNA(Sec) accommodates and peptide bond synthesis occurs .

Beyond selenocysteine incorporation, SECISBP2 also plays a distinct role in stabilizing certain selenoprotein mRNAs. Research has demonstrated that for several selenoproteins, loss of SECISBP2 resulted in greatly diminished mRNA levels, while translational activity and selenocysteine incorporation efficiency remained unaffected in the remaining RNA .

What applications are SECISBP2 antibodies validated for?

SECISBP2 antibodies have been validated for multiple applications in molecular and cellular biology research. The applications include:

When selecting an antibody for your specific application, consider the validation status for your particular experimental system. The Proteintech SECISBP2 antibody (12798-1-AP), for example, has been cited in multiple publications for applications including knockdown/knockout validation, Western blotting, immunohistochemistry, and immunofluorescence .

How should I validate the specificity of SECISBP2 antibodies?

Validating antibody specificity is crucial for obtaining reliable research results. For SECISBP2 antibodies, implement the following validation strategies:

• Genetic Validation:

  • Use SECISBP2 knockout or knockdown models as negative controls

  • Verify signal reduction/elimination in these models compared to wild-type samples

  • Multiple publications have utilized this approach with SECISBP2 antibodies

• Western Blot Analysis:

  • Confirm detection of the expected 95 kDa band (predicted molecular weight)

  • Run multiple cell/tissue types to verify consistent banding patterns

  • Include positive controls from validated cell lines (HeLa, MCF7, BT-474, 293T cells)

• Isoform Consideration:

  • Be aware that SECISBP2 exists in multiple splice variants, including a mitochondrial isoform (mtSECISBP2)

  • Determine which isoforms your antibody should detect based on the epitope location

  • For comprehensive analysis, consider using antibodies recognizing different epitopes

• Cross-reactivity Assessment:

  • Test for potential cross-reactivity with the related SECISBP2L protein, especially in neural tissue where SECISBP2L is specifically expressed in oligodendrocytes

  • Include appropriate cell types known to express only one of these proteins

• Functional Correlation:

  • Correlate SECISBP2 detection with selenoprotein expression levels

  • In disease models with SECISBP2 mutations, verify that antibody signals correlate with functional defects in selenoprotein synthesis

These validation steps ensure reliable detection of SECISBP2 in your experimental system and minimize the risk of misinterpreting results due to antibody non-specificity.

What are the optimal protocols for Western blot analysis of SECISBP2?

For optimal Western blot detection of SECISBP2, follow these methodology recommendations:

• Sample Preparation:

  • Use universal immunoprecipitation buffer (UIP) for cell lysis as described in published protocols

  • Include protease inhibitors to prevent degradation

  • For tissues, homogenize thoroughly in cold lysis buffer

• Gel Electrophoresis:

  • Use 4-12% gradient gels for optimal resolution of the 95 kDa SECISBP2 protein

  • Load adequate protein (20-50 μg total protein per lane)

  • Include molecular weight markers covering the 70-130 kDa range

• Transfer and Blocking:

  • Transfer to PVDF membrane (as used in published protocols)

  • Block with 5% skimmed milk powder for 1 hour at room temperature

• Antibody Incubation:

  • Primary antibody: Use at 1:500-1:2000 dilution

  • Incubate overnight at 4°C

  • Wash thoroughly (3-5 times) with TBST

• Detection:

  • Use HRP-conjugated secondary antibodies

  • Develop using enhanced chemiluminescence (ECL) detection system

  • For quantitative analysis, consider digital imaging systems (e.g., LAS-3000 luminescent image analyzer as used in published work)

• Controls and Validation:

  • Include positive controls (HeLa, MCF7, BT-474, 293T cells, or mouse thymus tissue)

  • Use GAPDH or actin as loading controls

  • For isoform studies, consider the predicted molecular weights of specific variants

• Troubleshooting Common Issues:

  • High background: Increase washing steps or dilute antibody further

  • Weak signal: Increase protein loading or decrease antibody dilution

  • Multiple bands: May represent isoforms or post-translational modifications; verify with isoform-specific controls

Following these optimized protocols will enhance detection specificity and sensitivity when analyzing SECISBP2 expression in your experimental system.

How should I optimize immunofluorescence protocols for SECISBP2 detection?

For successful immunofluorescence detection of SECISBP2, follow these detailed optimization steps:

• Sample Preparation:

  • Fix cells with 4% paraformaldehyde in PBS for 30 minutes at room temperature

  • For tissue sections, fix overnight at 4°C in 4% paraformaldehyde/PBS, followed by 20% sucrose infusion and OCT embedding

  • Prepare cryosections at 14-18 μm thickness

• Permeabilization and Blocking:

  • Permeabilize with 0.1-0.5% Triton X-100 in PBS

  • Block with 3% horse serum in PBS as used in published protocols

  • Blocking for 1 hour at room temperature is typically sufficient

• Antibody Incubation:

  • Primary antibody: Use at 1:50-1:800 dilution range

  • For Abcam's antibody (ab210791), a 1:50 dilution has been verified

  • For Proteintech's antibody (12798-1-AP), a 1:200-1:800 dilution range is recommended

  • Incubate overnight at 4°C for optimal results

• Secondary Antibody and Counterstaining:

  • Use fluorophore-conjugated secondary antibodies (e.g., Alexa fluor 488)

  • Counterstain nuclei with DAPI

  • For mitochondrial colocalization studies, use MitoTracker CMXRos (50 nM)

• Mounting and Imaging:

  • Mount with ProLong Gold or similar anti-fade mounting medium

  • Use confocal microscopy for detailed subcellular localization analysis

  • For colocalization studies, acquire sequential channels to prevent bleed-through

• Controls and Validation:

  • Include primary antibody omission controls

  • For mitochondrial SECISBP2 isoform studies, confirm colocalization with mitochondrial markers

  • Consider using cells with known expression patterns (HeLa, HepG2) as positive controls

• Specialized Applications:

  • Isoform-Specific Detection: For detecting the mitochondrial isoform (mtSECISBP2), use double immunofluorescent procedures with mitochondrial markers

  • Stress Response Studies: Include appropriate stress markers in co-staining experiments

By optimizing these parameters for your specific cellular system, you can achieve reliable visualization of SECISBP2 subcellular localization and expression patterns.

How can I design experiments to distinguish between SECISBP2's roles in selenocysteine incorporation and mRNA stability?

Differentiating between SECISBP2's dual functions requires sophisticated experimental design. Based on published research approaches , implement the following strategy:

• Comparative Gene Knockout Strategy:

  • Generate conditional knockout models for both SECISBP2 and tRNA(Sec) (encoded by the TRSP gene)

  • Compare the effects on selenoprotein synthesis between these models

  • In tRNA(Sec) knockout, expect uniform loss of selenoprotein synthesis

  • In SECISBP2 knockout, expect gene-specific effects that reveal its differential roles

• Ribosome Profiling Analysis:

  • Measure ribosome density upstream and downstream of UGA-Sec codons

  • Compare profiles in wild-type, SECISBP2-knockout, and tRNA(Sec)-knockout conditions

  • tRNA(Sec) depletion should show consistent loss of ribosome density downstream of all UGA-Sec codons

  • SECISBP2 depletion will show variable effects on ribosome density that reflect its differential roles across selenoprotein mRNAs

• mRNA Stability Assessment:

  • Implement RNA-Seq and mRNA half-life measurements

  • Use actinomycin D chase experiments to measure decay rates

  • Compare decay kinetics between wild-type and SECISBP2-depleted conditions

  • For mRNAs where SECISBP2 primarily functions in stability, expect accelerated decay upon SECISBP2 depletion

• Translational Activity Measurement:

  • For selenoproteins with greatly diminished mRNA levels upon SECISBP2 loss, evaluate:
    a. Translational activity on remaining mRNA
    b. Selenocysteine incorporation efficiency

  • If these parameters remain unaffected despite reduced mRNA levels, this indicates separable functions of SECISBP2

• SECIS Element Manipulation:

  • Design reporter constructs with wild-type or mutated SECIS elements

  • Assess how specific mutations differentially affect mRNA stability versus selenocysteine incorporation

  • Identify SECIS features specifically involved in each function

Published research demonstrates that "for several selenoproteins in which loss of Secisbp2 resulted in greatly diminished mRNA levels, translational activity and Sec incorporation efficiency were shown to be unaffected on the remaining RNA," supporting distinct mechanistic roles for SECISBP2 .

What methodologies are available for studying SECISBP2 isoforms, particularly the mitochondrial variant?

Investigating SECISBP2 isoforms requires specialized approaches to distinguish between functionally distinct protein variants. Based on published methodologies , implement these research strategies:

• Isoform Identification and Characterization:

  • In silico Analysis: Use bioinformatics tools (e.g., PSORT II) to predict subcellular localization of potential isoforms

  • RT-PCR Analysis: Design primers spanning alternative splice junctions to identify isoform expression patterns

  • Sequencing Verification: Confirm alternative splicing events through cDNA sequencing

• Subcellular Localization Studies:

  • Co-localization Imaging:

    • For mtSECISBP2, use double immunofluorescent procedures with MitoTracker CMXRos (50 nM)

    • Counterstain nuclei with DAPI

    • Analyze using confocal microscopy

  • Subcellular Fractionation:

    • Isolate mitochondrial, cytosolic, and nuclear fractions

    • Perform Western blotting to detect isoform distribution

    • Verify fraction purity with compartment-specific markers

• Minigene-Based Splicing Analysis:

  • Construct minigenes containing the 5'-region of SECISBP2 that undergoes alternative splicing

  • Transfect into relevant cell types and analyze splicing patterns

  • Modulate splicing using antisense oligonucleotides (ASOs) to alter isoform ratios

• Functional Characterization:

  • Generate GFP-tagged isoform constructs for visualization and functional studies

  • Create isoform-specific knockdown/knockout models

  • Perform rescue experiments with individual isoforms to determine functional specificity

• Stress Response Analysis:

  • Examine how various cellular stressors affect isoform expression and localization

  • Investigate isoform-specific roles in coordinated stress responses

Research has revealed that human SECISBP2 exhibits a complex splicing pattern in its 5'-region, producing at least eight splice variants encoding five isoforms with varying N-terminal sequences . The mitochondrial isoform (mtSECISBP2) contains a specific mitochondrial targeting sequence that directs localization to mitochondria . These methodologies provide a framework for systematically investigating the expression, regulation, and function of different SECISBP2 isoforms.

How can I use SECISBP2 antibodies to investigate selenoprotein synthesis defects in disease models?

SECISBP2 antibodies are valuable tools for investigating selenoprotein synthesis defects in disease models. Based on published research approaches , implement this comprehensive strategy:

• Disease Model Selection and Characterization:

  • Human Patient Samples: Tissues/cells from individuals with thyroid hormone metabolism abnormalities

  • Animal Models: Conditional SECISBP2 knockout mice (similar to the SECISBP2L conditional knockout described in the literature)

  • Cell Culture Models: SECISBP2 knockdown/knockout cell lines or cells expressing disease-associated SECISBP2 mutations

• Multi-level Analysis Strategy:

  Protein Expression Analysis:

  • Western Blotting:

    • Quantify SECISBP2 levels using 1:500-1:2000 antibody dilution

    • Simultaneously examine key selenoproteins (e.g., SELENOP)

    • Compare expression patterns between disease and control samples

  Tissue/Cell Distribution:

  • Immunohistochemistry:

    • Examine tissue-specific expression patterns (1:200 dilution)

    • Look for altered distribution in disease tissues

    • Compare with selenoprotein localization

  Subcellular Localization:

  • Immunofluorescence:

    • Detect changes in subcellular localization (1:50-1:800 dilution)

    • Co-stain with organelle markers, especially for mitochondrial isoform studies

• Functional Correlation Studies:

  • Selenoprotein Activity Assays:

    • Measure activities of selenoenzymes (e.g., deiodinases)

    • Correlate with SECISBP2 expression/localization

  • Thyroid Hormone Metabolism:

    • Measure T3 and T4 levels using ELISA

    • Assess deiodinase activities

    • Link to SECISBP2 expression/function

• SECIS-Binding Analysis:

  • Luciferase Reporter Assays:

    • Test SECIS elements from different selenoproteins (e.g., Dio1, Dio2, Dio3)

    • Compare wild-type versus mutant SECISBP2 binding efficiency

    • Identify selenoprotein mRNAs most affected by specific mutations

• Case Study Application from Literature:
  Clinical studies have identified mutations in SECISBP2 in families presenting with abnormal thyroid hormone metabolism . These patients exhibited:

  • Lack of functional selenoenzyme deiodinase 2 (DIO2)

  • Dramatically reduced levels of selenoprotein P

  • Specific defects in selenoprotein synthesis

This comprehensive approach allows researchers to establish mechanistic links between SECISBP2 dysfunction, selenoprotein deficiencies, and resultant pathologies in disease settings.

How can I troubleshoot variable results when using SECISBP2 antibodies across different experimental systems?

When encountering inconsistent results with SECISBP2 antibodies across different cell types or experimental conditions, implement this systematic troubleshooting approach:

• Expression Level Variation Assessment:

  • Issue: SECISBP2 expression naturally varies between cell types

  • Solution:

    • Adjust protein loading amounts (higher for low-expressing cells)

    • Optimize antibody concentration for each cell type

    • Verify mRNA expression levels by qRT-PCR to confirm expected expression differences

• Isoform Expression Analysis:

  • Issue: Different cell types may express distinct SECISBP2 isoforms

  • Solution:

    • Verify antibody epitope location relative to alternatively spliced regions

    • Use RT-PCR with isoform-specific primers to profile variant expression

    • Consider alternative antibodies recognizing different epitopes

• Application-Specific Optimization:

  Application	Optimization Parameters
  Western Blot	- Extraction method: Try different lysis buffers - Antibody dilution: Test range from 1:500-1:2000 - Blocking agent: Compare BSA vs. milk - Gel type: Use 4-12% gradient gels for better resolution
  Immunofluorescence	- Fixation method: Standard is 4% paraformaldehyde - Permeabilization: Adjust Triton X-100 concentration - Antibody dilution: Test range from 1:50-1:800 - Antigen retrieval: May be necessary for some samples

• Cross-Reactivity Considerations:

  • Issue: Potential cross-reactivity with SECISBP2L or other related proteins

  • Solution:

    • Include knockout/knockdown controls

    • Be particularly cautious in neural tissues where SECISBP2L is expressed in oligodendrocytes

    • Use antibodies validated with genetic knockout controls

• Reference Cell Types from Literature:
  Successfully validated cell types include:

  • Western blot: HeLa, MCF7, BT-474, 293T cells, and mouse thymus

  • Immunofluorescence: HeLa and HepG2 cells

  • Immunohistochemistry: Human colon carcinoma and liver injury tissues

  Start with these validated systems when establishing new protocols.

• Experimental Consistency:

  • Process all comparative samples simultaneously

  • Maintain consistent sample preparation methods

  • Use internal controls for normalization

By systematically addressing these factors, you can identify the source of variability and establish reliable protocols for consistent SECISBP2 detection across different experimental systems.

What are the key differences between SECISBP2 and SECISBP2L, and how can I ensure antibody specificity?

SECISBP2 and SECISBP2L (SECISBP2-like) are related proteins with distinct tissue expression patterns and potentially overlapping functions. Understanding their differences and ensuring antibody specificity is crucial for accurate research:

• Biological Distinctions:

  Feature	SECISBP2	SECISBP2L
  Expression Pattern	Widely expressed across tissues	Specifically expressed in differentiating oligodendrocytes
  Function	General selenoprotein synthesis; mutations linked to thyroid hormone metabolism disorders	Essential for selenoprotein translation specifically in oligodendrocytes
  Disease Association	Abnormal thyroid hormone metabolism in humans with mutations	Essential for proper myelination in the central nervous system

• Ensuring Antibody Specificity:

  Epitope Selection:

  • Choose antibodies raised against regions with low sequence homology

  • Verify immunogen sequence details from antibody manufacturers

  • Consider epitopes outside conserved functional domains

  Validation Controls:

  • Use SECISBP2 knockout/knockdown samples as negative controls for SECISBP2 antibodies

  • Use SECISBP2L knockout/knockdown samples (e.g., from SECISBP2L conditional knockout mice ) as controls for SECISBP2L antibodies

  • Implement tissue-specific validation using oligodendrocyte-rich versus oligodendrocyte-poor regions

  Cell Type-Specific Validation:

  • Utilize oligodendrocyte markers (OLIG2, CC1, MAG, MBP ) in co-staining experiments

  • Compare staining patterns in oligodendrocyte-specific SECISBP2L knockout tissues

• Critical Experimental Approaches:

  Western Blot Differentiation:

  • Run samples side-by-side with both antibodies

  • Compare with recombinant protein standards if available

  • Look for size differences or differential expression patterns

  Immunofluorescence Specificity:

  • Perform double-labeling with cell type-specific markers

  • Use sequential conditional knockout models

  • Implement antibody pre-adsorption with recombinant proteins

This comprehensive approach ensures reliable distinction between these related proteins, preventing misinterpretation of experimental results due to antibody cross-reactivity.

Future directions in SECISBP2 antibody applications for selenium biology research

As the field of selenium biology continues to evolve, SECISBP2 antibodies will play increasingly important roles in unraveling the complexities of selenoprotein synthesis regulation. Future research directions should focus on:

• Integrative Multi-omics Approaches:

  • Combining SECISBP2 immunoprecipitation with RNA-Seq to comprehensively identify bound selenoprotein mRNAs

  • Correlating SECISBP2 binding patterns with selenoprotein translation efficiency

  • Integrating proteomic and transcriptomic data to build mechanistic models of selenoprotein synthesis regulation

• Disease-Specific Applications:

  • Investigating SECISBP2 dysfunction in additional disease contexts beyond thyroid disorders

  • Exploring the role of SECISBP2 in neurodegenerative diseases, cancer, and inflammatory conditions

  • Developing diagnostic applications for detecting SECISBP2 defects in patient samples

• Isoform-Specific Functional Analysis:

  • Creating isoform-selective antibodies for targeted studies of mtSECISBP2 and other variants

  • Investigating the distinct roles of SECISBP2 isoforms in different cellular compartments

  • Examining isoform expression changes during development and in response to environmental challenges

• Therapeutic Target Validation:

  • Using SECISBP2 antibodies to validate potential therapeutic approaches targeting selenoprotein synthesis

  • Developing screening assays for compounds that modulate SECISBP2 function

  • Investigating SECISBP2 as a potential biomarker for disease states or treatment response