SPTBN1 Antibody, Biotin conjugated

What is SPTBN1 and what are its key biological functions?

SPTBN1 (Spectrin Beta, Non-Erythrocytic 1) is a cytoskeletal protein also known as Beta-II spectrin, Fodrin beta chain, or SPTB2. This protein plays several critical biological roles, particularly in membrane stability and cellular architecture. SPTBN1 is primarily involved in secretion processes and interacts with calmodulin in a calcium-dependent manner, making it a key candidate for calcium-dependent movement of the cytoskeleton at the membrane . The protein has a calculated molecular weight of 275 kDa and is encoded by the gene with ID 6711 .

SPTBN1 plays a particularly crucial role in central nervous system development and function . Recent research has also identified potential tumor suppressor functions, as reduced expression of SPTBN1 correlates with shorter survival rates in patients with hepatocellular cancer, pancreatic cancer, and other gastrointestinal tract malignancies . This suggests that SPTBN1 may have applications in both neuroscience and cancer research contexts.

What are the benefits of using biotin-conjugated SPTBN1 antibodies compared to unconjugated versions?

Biotin-conjugated SPTBN1 antibodies offer several significant advantages for research applications:

• Enhanced sensitivity: The biotin-avidin/streptavidin system provides signal amplification due to the multiple biotin molecules that can bind to each avidin/streptavidin molecule, substantially improving detection sensitivity in assays like ELISA .

• Flexible detection systems: Biotin-conjugated antibodies can be detected using various avidin/streptavidin conjugates (HRP, fluorophores, gold particles), allowing researchers to adapt to different visualization methods without changing the primary antibody .

• Reduced background: When properly optimized, biotin-conjugated antibodies can reduce non-specific binding compared to directly labeled antibodies, especially in multicolor immunostaining protocols.

• Versatility across applications: Biotin-conjugated SPTBN1 antibodies have demonstrated effectiveness in ELISA applications with recommended dilutions of 1:500-1:1000, making them suitable for various detection methods .

The biotin conjugation is achieved through protein G purification and careful conjugation chemistry to maintain antibody specificity while adding the biotin tag .

How do I select between polyclonal and monoclonal SPTBN1 antibodies for my research?

The choice between polyclonal and monoclonal SPTBN1 antibodies depends on your specific research objectives:

Polyclonal SPTBN1 Antibodies:

• Recognize multiple epitopes on the SPTBN1 protein, potentially providing stronger signals, especially for proteins expressed at low levels

• Available as rabbit-derived polyclonal antibodies with reactivity to human and mouse samples

• More tolerant of minor changes in protein structure or slight protein denaturation

• Particularly useful for initial protein characterization and applications like Western blotting and immunohistochemistry

• Examples include catalog ABIN6148407 (unconjugated) and biotin-conjugated options for ELISA applications

Monoclonal SPTBN1 Antibodies:

• Recognize a single epitope with high specificity

• Provide more consistent lot-to-lot reproducibility

• Available as rabbit recombinant monoclonal antibodies (e.g., EPR5869) with reactivity to human, mouse, and rat samples

• Best for applications requiring high specificity and reproducibility

• Particularly valuable for quantitative applications and detecting specific protein isoforms

For most exploratory studies, polyclonal antibodies provide stronger signals across multiple applications, while monoclonal antibodies excel in standardized protocols requiring consistent reagents over extended research periods .

What are the validated applications for SPTBN1 biotin-conjugated antibodies?

SPTBN1 biotin-conjugated antibodies have been validated for the following applications:

While biotin-conjugated SPTBN1 antibodies are primarily validated for ELISA applications, the unconjugated versions of the same antibody clones have demonstrated effectiveness in Western blotting, immunohistochemistry, and immunoprecipitation . If considering alternative applications beyond ELISA, pilot experiments to validate the biotin-conjugated version for your specific experimental system are recommended.

How should I optimize SPTBN1 biotin-conjugated antibody dilutions for ELISA?

Optimizing dilutions for SPTBN1 biotin-conjugated antibodies in ELISA requires a systematic approach:

• Initial titration experiment:

  • Begin with the manufacturer's recommended dilution range (1:500-1:1000)

  • Prepare a dilution series (e.g., 1:250, 1:500, 1:1000, 1:2000)

  • Run parallel assays with positive and negative controls

• Signal-to-noise ratio assessment:

  • Calculate the ratio between specific signal and background for each dilution

  • The optimal dilution provides the highest signal-to-noise ratio, not necessarily the strongest absolute signal

• Cross-reactivity evaluation:

  • Test the antibody against related proteins to ensure specificity

  • For SPTBN1, verify no cross-reactivity with other spectrin family members

• Validation across sample types:

  • Different sample preparations may require adjusted dilutions

  • Human tissue samples may require different optimization than cell lines

• Blocking optimization:

  • When using biotin-conjugated antibodies, special attention to blocking endogenous biotin is critical

  • Test different blocking reagents to minimize background while maintaining specific signal

Document your optimization process thoroughly, as the optimal dilution may vary based on the specific ELISA format, detection system, and sample type .

What sample preparation methods are most effective for SPTBN1 detection?

Effective sample preparation for SPTBN1 detection varies by application and tissue type:

For Western Blotting:

• SPTBN1 is a large protein (275 kDa), requiring careful gel selection (typically 6-8% gels or gradient gels)

• Use fresh tissue samples when possible, as SPTBN1 may be susceptible to proteolytic degradation

• Include protease inhibitors in lysis buffers

• Positive control samples include mouse brain, kidney, and lung tissues, which have demonstrated detectable SPTBN1 expression

For Immunohistochemistry:

• Antigen retrieval is critical; recommended methods include:

  • TE buffer pH 9.0 (preferred)

  • Alternative: citrate buffer pH 6.0

• SPTBN1 detection has been validated in multiple human tissues including liver cancer tissue, lung tissue, kidney tissue, pancreas cancer tissue, and normal colon

For ELISA with Biotin-Conjugated Antibodies:

• Carefully block endogenous biotin, particularly important in tissues like liver, kidney, and brain

• Consider pre-treatment with avidin/biotin blocking kits

• For recombinant proteins, verify that the expression system doesn't interfere with detection

Regardless of application, always verify SPTBN1 detection using positive control samples that have established expression, such as human or mouse brain tissues .

What are common challenges when using biotin-conjugated SPTBN1 antibodies and how can they be addressed?

Several challenges can arise when working with biotin-conjugated SPTBN1 antibodies:

High Background Signal:

• Cause: Endogenous biotin in samples (especially prevalent in liver, brain, and kidney tissues)

• Solution: Implement an avidin/biotin blocking step before antibody incubation

• Alternative: Use specialized blocking reagents designed for biotin-streptavidin systems

Weak or Absent Signal:

• Cause: Insufficient antibody concentration or protein denaturation

• Solution: Titrate antibody concentration and optimize antigen retrieval conditions

• For SPTBN1 specifically, ensure protein transfer conditions are optimized for high molecular weight proteins (275 kDa)

Non-specific Binding:

• Cause: Insufficient blocking or cross-reactivity

• Solution: Extend blocking time or try alternative blocking reagents

• Validate specificity using SPTBN1 knockout or knockdown samples as negative controls

Biotin Conjugation Interference:

• Cause: The biotin label may occasionally mask the antibody's epitope binding region

• Solution: Compare results with unconjugated versions of the same antibody

• Consider alternative detection methods if consistent issues arise

For reproducible results with biotin-conjugated SPTBN1 antibodies, maintain consistent experimental conditions, particularly incubation times and temperatures, as these significantly impact signal strength and specificity .

How can I validate the specificity of SPTBN1 biotin-conjugated antibodies?

Validating the specificity of SPTBN1 biotin-conjugated antibodies involves multiple complementary approaches:

• Positive and negative control samples:

  • Positive controls: Human liver cancer tissue, lung tissue, kidney tissue, pancreas cancer tissue, and normal colon have demonstrated SPTBN1 expression

  • Negative controls: Include samples known to lack SPTBN1 expression or SPTBN1 knockout/knockdown models

• Peptide competition assay:

  • Pre-incubate the antibody with the immunizing peptide (amino acids 2096-2256 of human SPTBN1 for most biotin-conjugated antibodies)

  • A specific antibody will show reduced or eliminated signal when the immunizing peptide blocks binding sites

• Multiple antibody validation:

  • Compare results using different antibodies targeting distinct SPTBN1 epitopes

  • Consistent detection patterns across antibodies increases confidence in specificity

  • Compare with unconjugated versions of the same antibody clone

• Western blot molecular weight verification:

  • SPTBN1 should appear at approximately 275 kDa

  • Absence of non-specific bands supports antibody specificity

• Cross-species reactivity assessment:

  • Most SPTBN1 biotin-conjugated antibodies show reactivity to human samples, with some also reactive to mouse samples

  • Expected cross-reactivity should match manufacturer specifications

Document all validation steps thoroughly, as this information strengthens the reliability of subsequent experimental findings and may be required for publication .

What detection systems work best with biotin-conjugated SPTBN1 antibodies?

For optimal results with biotin-conjugated SPTBN1 antibodies, several detection systems can be employed, each with specific advantages:

Streptavidin-HRP Systems:

• Provide excellent sensitivity for ELISA and Western blot applications

• Typically used with substrates like TMB (for ELISA) or ECL (for Western blots)

• Advantage: High sensitivity due to the strong biotin-streptavidin interaction

• Recommended for quantitative ELISA applications of SPTBN1

Streptavidin-Fluorophore Conjugates:

• Enable fluorescent detection for microscopy applications

• Options include streptavidin-FITC, streptavidin-Cy3, or streptavidin-Alexa Fluor conjugates

• Advantage: Compatible with multiplex staining to co-localize SPTBN1 with other proteins

• Particularly useful for examining SPTBN1's interaction with calmodulin or cytoskeletal components

Avidin-Biotin Complex (ABC) Systems:

• Amplify signal through the formation of avidin-biotin complexes

• Particularly valuable for detecting low-abundance proteins or in tissues with weak expression

• Advantage: Enhanced sensitivity compared to direct detection methods

• Useful for IHC applications examining SPTBN1 in cancer tissues

Tyramide Signal Amplification (TSA):

• Combines biotin-streptavidin binding with tyramide amplification

• Provides exceptional sensitivity for detecting low levels of SPTBN1

• Advantage: Can improve signal detection by 10-100 fold over conventional methods

• Valuable for examining SPTBN1 expression in samples where protein levels are minimal

When selecting a detection system, consider your specific application goals, required sensitivity, and instrumentation availability. For quantitative applications, maintain consistent detection systems across experiments to ensure comparable results .

How can SPTBN1 biotin-conjugated antibodies be used to investigate its tumor suppressor role?

SPTBN1 biotin-conjugated antibodies offer valuable tools for investigating the protein's emerging role as a tumor suppressor, particularly in gastrointestinal malignancies:

Tissue Microarray Analysis:

• Use biotin-conjugated SPTBN1 antibodies in high-throughput ELISA or immunohistochemical analyses of tumor tissue microarrays

• Correlate SPTBN1 expression levels with clinical outcomes and patient survival data

• This approach enables quantitative assessment of the relationship between SPTBN1 expression and tumor progression

Mechanistic Studies in Cancer Models:

• Employ ELISA with biotin-conjugated SPTBN1 antibodies to quantify protein expression changes during:

  • Cancer progression stages

  • Response to therapeutic interventions

  • Genetic manipulation of oncogenic pathways

• The high sensitivity of biotin-streptavidin detection systems is particularly valuable for detecting subtle expression changes

Prognostic Marker Validation:

• Develop standardized ELISA protocols using biotin-conjugated SPTBN1 antibodies for potential clinical applications

• Establish quantitative thresholds correlating with patient outcomes

• SPTBN1 expression has been specifically linked to survival in hepatocellular cancer, pancreatic cancer, and other gastrointestinal tract malignancies

Pathway Analysis:

• Use pull-down assays with biotin-conjugated SPTBN1 antibodies to identify binding partners in normal versus cancerous tissues

• Investigate how SPTBN1 interactions change during malignant transformation

• Elucidate the molecular mechanisms underlying its tumor suppressor function

This research direction is particularly promising given the documented correlation between reduced SPTBN1 expression and shorter survival in multiple cancer types, suggesting potential diagnostic and prognostic applications .

What considerations are important when using SPTBN1 biotin-conjugated antibodies for cytoskeletal dynamics studies?

When investigating SPTBN1's role in cytoskeletal dynamics with biotin-conjugated antibodies, researchers should consider several specialized factors:

Preservation of Cytoskeletal Architecture:

• Fixation method significantly impacts SPTBN1 detection in cytoskeletal structures

• For immunofluorescence studies, paraformaldehyde fixation (4%, 10-15 minutes) typically preserves SPTBN1's native conformation and interactions

• Avoid methanol fixation which can disrupt spectrin's association with other cytoskeletal components

Calcium-Dependent Interactions:

• SPTBN1 interacts with calmodulin in a calcium-dependent manner

• Consider parallel experiments with calcium chelators (e.g., EGTA) to distinguish calcium-dependent from calcium-independent interactions

• Use biotin-conjugated SPTBN1 antibodies in conjunction with fluorescently labeled calmodulin to study co-localization dynamics

Membrane Association Studies:

• SPTBN1 plays a role in calcium-dependent movement of the cytoskeleton at the membrane

• When designing experiments to study this function:

  • Include membrane markers in co-staining protocols

  • Consider subcellular fractionation followed by ELISA to quantify membrane-associated versus cytosolic SPTBN1

  • Employ super-resolution microscopy for detailed visualization of membrane-cytoskeleton interfaces

Central Nervous System Applications:

• Given SPTBN1's critical role in CNS development and function :

  • Use biotin-conjugated antibodies in developmental studies to track expression changes

  • Compare SPTBN1 localization across different neural cell types

  • Investigate potential role in neurological disorders through comparative tissue analysis

For all cytoskeletal studies, consider the large size of SPTBN1 (275 kDa) when designing experimental approaches, as this may affect protein extraction efficiency, antibody accessibility, and detection sensitivity .

How can I combine SPTBN1 biotin-conjugated antibodies with other detection methods for multiplexed analysis?

Multiplexed analysis combining SPTBN1 biotin-conjugated antibodies with other detection methods enables comprehensive investigation of complex biological processes:

Multiplex Immunofluorescence Strategies:

• When combining biotin-conjugated SPTBN1 antibodies with other fluorescent probes:

  • Use streptavidin conjugated to spectrally distinct fluorophores (e.g., Cy5, Alexa 647) to avoid spectral overlap with common fluorophores

  • Apply sequential staining protocols to prevent cross-reactivity

  • Include appropriate controls to verify absence of antibody cross-reactivity or fluorophore bleed-through

Sequential Chromogenic IHC:

• For multiplexed chromogenic detection:

  • Start with biotin-conjugated SPTBN1 antibody detection using one chromogen

  • Perform antibody stripping or inactivation

  • Continue with subsequent antibody staining using different chromogens

  • This approach allows visualization of multiple proteins on the same tissue section

Combined IF-FISH Approaches:

• Integrate SPTBN1 protein detection with fluorescence in situ hybridization:

  • Detect SPTBN1 protein using biotin-conjugated antibodies and streptavidin-fluorophore

  • Combine with FISH probes for SPTBN1 gene or related genes

  • This enables simultaneous analysis of protein expression and genetic alterations

Mass Cytometry Applications:

• For high-dimensional analysis:

  • Conjugate metal isotopes to streptavidin for detection of biotin-conjugated SPTBN1 antibodies

  • Combine with dozens of other metal-labeled antibodies

  • Analyze using CyTOF or Imaging Mass Cytometry for single-cell protein expression profiling

For successful multiplexed experiments, carefully optimize each step of the protocol, including fixation conditions, antigen retrieval methods, blocking procedures, and detection systems. Proper controls should include single-stain controls, isotype controls, and fluorescence-minus-one controls to ensure accurate interpretation of results .

What quantitative approaches can be used with SPTBN1 biotin-conjugated antibodies for expression analysis?

Several quantitative approaches can be employed with SPTBN1 biotin-conjugated antibodies to obtain reliable expression data:

Quantitative ELISA:

• Develop standard curves using recombinant SPTBN1 protein at known concentrations

• Apply biotin-conjugated SPTBN1 antibodies at optimized dilutions (1:500-1:1000)

• Implement four-parameter logistic regression for accurate concentration determination

• This method is particularly valuable for comparing SPTBN1 expression across different tissue types or experimental conditions

Densitometric Analysis:

• For Western blots using biotin-conjugated SPTBN1 antibodies:

  • Include recombinant SPTBN1 standards for calibration

  • Normalize SPTBN1 signal to appropriate loading controls

  • Use specialized software for band intensity quantification

  • Account for the high molecular weight of SPTBN1 (275 kDa) when optimizing transfer conditions

Automated Tissue Analysis:

• For immunohistochemistry applications:

  • Apply digital pathology algorithms to quantify SPTBN1 staining

  • Establish scoring systems based on staining intensity and percentage of positive cells

  • Implement machine learning approaches for consistent analysis across large sample sets

  • This approach has been successfully applied to SPTBN1 analysis in cancer tissues

Flow Cytometry:

• For cellular expression analysis:

  • Use permeabilized cells with biotin-conjugated SPTBN1 antibodies and fluorescent streptavidin

  • Establish quantitative gates based on control samples

  • Correlate SPTBN1 expression with cell cycle phase or other cellular parameters

  • Consider using fluorescence standards for absolute quantification

For all quantitative applications, maintaining consistent experimental conditions is essential, including antibody lot, incubation times, detection reagents, and instrument settings. Regular inclusion of internal standards and quality controls ensures reliable, comparable results across experiments .