TM4SF20 Antibody, Biotin conjugated

What is TM4SF20 and why is it a target for antibody development?

TM4SF20 (Transmembrane 4 L Six Family Member 20) is a human protein encoded by the TM4SF20 gene (GeneID: 79853). It belongs to the transmembrane 4 L6 family of proteins characterized by four transmembrane domains. This protein has gained research interest due to its potential roles in cellular signaling and membrane organization. The development of specific antibodies against TM4SF20 enables researchers to study its expression, localization, and function in various physiological and pathological contexts. Recent studies have associated TM4SF20 deletions with pediatric language impairment and brain abnormalities, highlighting its significance in neurodevelopmental research .

What are the key specifications of the Biotin-conjugated TM4SF20 antibody?

The Biotin-conjugated TM4SF20 antibody is a polyclonal antibody raised in rabbits against the amino acid region 114-184 of the human TM4SF20 protein . It demonstrates specificity for human TM4SF20 and has been validated for ELISA applications . The antibody is provided in liquid form with >95% purity, purified using Protein G affinity chromatography . It is prepared in a buffer containing 0.01 M PBS (pH 7.4), 0.03% Proclin-300, and 50% Glycerol . This antibody is strictly for research use only and not intended for diagnostic, therapeutic, or cosmetic applications .

How does Biotin conjugation enhance the utility of the TM4SF20 antibody?

Biotin conjugation offers several methodological advantages for TM4SF20 detection. The biotin molecule forms an exceptionally strong non-covalent bond with streptavidin or avidin proteins, providing a robust platform for signal amplification in various immunoassays. This conjugation allows for flexible detection systems as researchers can employ different streptavidin-conjugated reporter molecules (e.g., HRP, fluorophores) without needing multiple directly labeled primary antibodies. The biotin-streptavidin system typically yields enhanced sensitivity compared to conventional direct detection methods, making it particularly valuable for detecting low-abundance proteins like TM4SF20. Additionally, the biotin tag minimally affects antibody binding properties while providing consistent and reproducible signal generation across experimental platforms .

What validated applications are supported by the Biotin-conjugated TM4SF20 antibody?

The primary validated application for the Biotin-conjugated TM4SF20 antibody is ELISA (Enzyme-Linked Immunosorbent Assay) . The antibody has been specifically optimized for quantitative sandwich enzyme immunoassay techniques, which are widely used for detecting and quantifying specific proteins in complex biological samples . While ELISA remains the primary validated application, related TM4SF20 antibodies (non-biotin conjugated) have demonstrated utility in additional applications including Western Blot (WB), Immunofluorescence/Immunocytochemistry (IF/ICC), and Immunohistochemistry (IHC) . Researchers interested in extending applications beyond ELISA should perform thorough validation studies to confirm antibody performance in their specific experimental systems.

What is the recommended methodology for determining optimal working dilutions of this antibody?

Determining optimal working dilutions for the Biotin-conjugated TM4SF20 antibody requires systematic titration experiments. The manufacturer guidelines indicate that optimal dilutions/concentrations should be determined by the end-user based on their specific experimental conditions . A recommended approach involves:

• Preparing a series of antibody dilutions (typically ranging from 1:100 to 1:10,000)

• Testing these dilutions against both positive controls (samples known to express TM4SF20) and negative controls (samples lacking TM4SF20 expression)

• Analyzing signal-to-noise ratios to identify the dilution that provides maximum specific signal with minimal background

• Validating the selected dilution across multiple independent experiments to ensure reproducibility

For ELISA applications, researchers should follow the standard sandwich ELISA procedure outlined in the kit protocols, which typically involves:

• Pre-coating microplates with capture antibody

• Adding standards and samples

• Incubating with the Biotin-conjugated detection antibody

• Adding streptavidin-HRP

• Developing with substrate solution

• Measuring optical density at 450nm with correction at 570nm or 630nm

What are the critical storage and handling considerations to maintain antibody performance?

Proper storage and handling are essential for maintaining the functional integrity of the Biotin-conjugated TM4SF20 antibody. The recommended storage conditions include:

• Aliquoting the antibody upon receipt to minimize freeze-thaw cycles

• Storing at -20°C or -80°C for long-term preservation

• Protecting from light exposure, as biotin conjugates are photosensitive

• Avoiding repeated freeze-thaw cycles, which can compromise antibody activity

When working with the antibody:

• Thaw aliquots completely but gently before use

• Keep on ice during experimental procedures

• Return unused portions to storage promptly

• Consider the shelf-life of the antibody (typically 12 months from date of receipt when properly stored)

• Be aware that the antibody contains ProClin, which is classified as a hazardous substance requiring appropriate handling precautions

How does the polyclonal nature of this antibody impact experimental outcomes and interpretation?

The polyclonal nature of the Biotin-conjugated TM4SF20 antibody carries specific methodological implications that researchers should consider when designing experiments and interpreting results. Unlike monoclonal antibodies that recognize a single epitope, this polyclonal antibody contains a heterogeneous mixture of immunoglobulins that recognize multiple epitopes within the 114-184 amino acid region of TM4SF20 . This characteristic provides both advantages and limitations:

Advantages:

• Enhanced sensitivity due to binding of multiple epitopes per target molecule

• Greater tolerance to minor changes in the antigen (from denaturation or fixation)

• Improved signal strength, particularly beneficial for detecting low-abundance proteins

Limitations:

• Potential for batch-to-batch variation, necessitating consistent validation

• Possible cross-reactivity with structurally similar proteins

• More complex epitope mapping compared to monoclonal antibodies

Researchers should implement appropriate controls to account for these factors, including isotype controls, pre-absorption controls with the immunizing peptide, and validation across multiple detection methods when possible .

What controls should be included when using this antibody in ELISA-based detection systems?

A robust experimental design for ELISA using the Biotin-conjugated TM4SF20 antibody should include multiple controls to ensure data reliability:

• Standard Curve Controls:

  • Include a full range of recombinant TM4SF20 standards (typically 0.32-20 ng/mL) to establish quantitative relationships

  • Prepare standards in the same matrix as experimental samples when possible

• Negative Controls:

  • Buffer-only wells (no antigen)

  • Samples known not to express TM4SF20

  • Secondary antibody-only wells (omitting the primary antibody)

• Positive Controls:

  • Known concentration of recombinant TM4SF20 protein

  • Samples with validated TM4SF20 expression

• Specificity Controls:

  • Pre-absorption with immunizing peptide to confirm binding specificity

  • Competitive inhibition tests with unlabeled antibody

• Technical Controls:

  • Duplicate or triplicate measurements for all samples and standards

  • Inter-assay control samples to monitor plate-to-plate variation

  • Dilution linearity samples to verify detection across concentration ranges

The TM4SF20 ELISA kit demonstrates high sensitivity with a detection range of 0.32-20 ng/mL and a minimum detectable dose typically less than 0.156 ng/mL . Intra-plate precision shows CV<10%, while inter-plate precision shows CV<15% .

How should samples be prepared to optimize antibody performance in different experimental systems?

Sample preparation significantly impacts antibody performance and result reliability. For optimal results with the Biotin-conjugated TM4SF20 antibody:

For ELISA Applications:

• Serum/Plasma Samples:

  • Collect using standard protocols with appropriate anticoagulants

  • Centrifuge at 1000×g for 15 minutes within 30 minutes of collection

  • If not assayed immediately, aliquot and store samples at ≤-20°C

  • Avoid repeated freeze-thaw cycles

  • Consider dilution series to establish optimal working concentration (recovery rates in serum samples average 88%, with a range of 86-95%)

• Cell Culture Supernatants:

  • Centrifuge at 300×g for 10 minutes to remove debris

  • Evaluate need for concentration if target protein levels are low

  • Account for media components that might interfere with detection

  • Anticipate recovery rates averaging 84% (range 81-92%)

• Tissue Homogenates:

  • Homogenize tissue in PBS with protease inhibitors

  • Sonicate or freeze-thaw to disrupt cell membranes

  • Centrifuge at 5000×g for 10 minutes

  • Collect supernatant for analysis

The linearity of detection varies by sample type. For cell culture media, linearity ranges from 80-117% of expected values across dilutions of 1:2 to 1:16. For serum samples, linearity ranges from 80-118% across the same dilution range .

What are common technical challenges encountered when using this antibody, and how can they be addressed?

Researchers working with the Biotin-conjugated TM4SF20 antibody may encounter several technical challenges that can be systematically addressed:

• High Background Signal:

  • Potential causes: Insufficient blocking, cross-reactivity, or excessive antibody concentration

  • Solutions: Optimize blocking conditions (duration, blocking agent), increase wash stringency, titrate antibody to lower concentration, pre-absorb with non-specific proteins

• Weak or Absent Signal:

  • Potential causes: Insufficient antigen in sample, antibody degradation, interference from sample components

  • Solutions: Concentrate samples, verify antibody activity with positive controls, minimize freeze-thaw cycles, use freshly prepared reagents

• Poor Reproducibility:

  • Potential causes: Inconsistent technique, reagent instability, sample heterogeneity

  • Solutions: Standardize protocols, prepare larger batches of working dilutions, increase technical replicates

• Non-Linear Standard Curve:

  • Potential causes: Improper standard preparation, plate reader limitations, inappropriate curve fitting

  • Solutions: Verify standard quality, ensure proper plate reader settings, use four-parameter logistic (4-PL) curve-fit as recommended for data analysis

The antibody demonstrates minimal cross-reactivity with TM4SF20 analogues, but researchers should remain aware that full cross-reactivity profiles may not be completely characterized .

How can researchers validate antibody specificity for their particular experimental system?

Validating the specificity of the Biotin-conjugated TM4SF20 antibody in a particular experimental system is crucial for ensuring reliable results. Comprehensive validation approaches include:

• Genetic Validation:

  • Compare antibody reactivity in wild-type versus TM4SF20 knockdown/knockout models

  • Test in cell lines with confirmed differential expression of TM4SF20

  • Consider complementary approaches like gene silencing with siRNA followed by antibody testing

• Biochemical Validation:

  • Competitive inhibition assays with the immunizing peptide (amino acids 114-184 of TM4SF20)

  • Immunoprecipitation followed by mass spectrometry identification

  • Orthogonal detection methods (e.g., using multiple antibodies targeting different epitopes)

• Technical Validation:

  • Analysis of dilution linearity to confirm dose-dependent detection

  • Spike-and-recovery experiments in the specific sample matrix being tested

  • Comparison of results across multiple lots of the antibody, if available

• Alternative Methods Comparison:

  • Correlation with mRNA expression levels detected by qPCR

  • Comparison with other commercial antibodies targeting TM4SF20

  • Validation through recombinant expression systems with tagged TM4SF20

Researchers should document validation results thoroughly and include appropriate controls in all experiments to support the specificity claims in their specific experimental context .

What considerations are important when using this antibody in multiplexed detection systems?

When incorporating the Biotin-conjugated TM4SF20 antibody into multiplexed detection systems, researchers should address several methodological considerations:

• Biotin-Streptavidin System Compatibility:

  • Ensure other detection reagents don't compete for streptavidin binding

  • Consider sequential detection if using multiple biotin-conjugated antibodies

  • Verify that endogenous biotin in samples doesn't interfere with detection

• Cross-Reactivity Assessment:

  • Test for potential cross-reactivity with other targets in the multiplex panel

  • Perform single-plex controls alongside multiplex experiments

  • Consider pre-absorption with related proteins if cross-reactivity is observed

• Signal Optimization:

  • Balance signal intensities across all analytes in the panel

  • Optimize antibody concentrations independently for multiplex applications

  • Consider differential dye labeling of streptavidin to distinguish signals

• Data Analysis Considerations:

  • Account for spectral overlap when using multiple fluorescent reporters

  • Implement appropriate compensation controls

  • Validate quantification across the dynamic range for each analyte

• Sample Considerations:

  • Ensure compatible sample preparation methods for all targets

  • Evaluate potential matrix effects specific to multiplex systems

  • Consider the impact of sample dilution on detection of targets with different abundance levels

Researchers should perform extensive validation of multiplex systems, comparing results with single-plex assays to ensure that multiplexing doesn't compromise detection sensitivity or specificity for TM4SF20 .

What is currently understood about TM4SF20's biological function, and how might this antibody contribute to functional studies?

While TM4SF20's precise biological functions remain under investigation, emerging evidence suggests roles in cellular development and neurobiology:

TM4SF20 belongs to the tetraspanin superfamily, which typically functions in membrane compartmentalization and organization of molecular complexes. Current research indicates that TM4SF20 may be involved in:

• Neurodevelopmental Processes: Genetic studies have linked TM4SF20 deletions to pediatric language impairment and white matter hyperintensities in the brain, suggesting roles in neurodevelopment and potentially myelination .

• Cell Signaling: As a transmembrane protein, TM4SF20 likely participates in signal transduction pathways, though specific mechanisms remain to be elucidated.

• Membrane Organization: Like other tetraspanin family members, TM4SF20 may contribute to the formation of tetraspanin-enriched microdomains (TEMs) that organize membrane proteins and regulate their functions.

The Biotin-conjugated TM4SF20 antibody can advance functional studies through:

• Quantitative analysis of TM4SF20 expression levels in different tissues and cell types

• Immunoprecipitation studies to identify protein-protein interactions

• Immunocytochemistry to determine subcellular localization

• Analysis of expression changes under various physiological and pathological conditions

Researchers can leverage the specificity for amino acids 114-184 to target a functionally relevant domain of the protein and monitor expression patterns in relation to cellular functions .

How can this antibody be applied in studying TM4SF20-associated disorders and pathologies?

The Biotin-conjugated TM4SF20 antibody offers valuable research applications for investigating TM4SF20-associated disorders:

• Neurodevelopmental Disorders:

  • Given the association between TM4SF20 deletions and pediatric language impairment, the antibody can help quantify TM4SF20 expression in neuronal tissues and cell types

  • Studies could compare TM4SF20 protein levels between patients with language impairment and healthy controls

  • The antibody could facilitate investigation of TM4SF20's role in white matter development, as abnormalities in white matter have been observed in patients with TM4SF20 deletions

• Methodological Approaches:

  • ELISA-based screening of patient samples to correlate TM4SF20 levels with clinical phenotypes

  • Tissue microarray analysis to examine expression patterns across brain regions

  • Correlation of protein expression with genetic variations in TM4SF20

• Research Model Applications:

  • Characterization of TM4SF20 expression in animal models of neurodevelopmental disorders

  • Analysis of expression changes following genetic manipulation of TM4SF20

  • Investigation of compensatory mechanisms in TM4SF20 deletion carriers

The high sensitivity of the antibody (detection limit <0.156 ng/mL) makes it particularly suitable for detecting potentially subtle changes in TM4SF20 expression that might be associated with pathological conditions .

What emerging techniques might enhance the utility of this antibody in cutting-edge research applications?

Several emerging techniques could extend the research applications of the Biotin-conjugated TM4SF20 antibody:

• Single-Cell Protein Analysis:

  • Integration with mass cytometry (CyTOF) to analyze TM4SF20 expression at single-cell resolution

  • Application in microfluidic antibody-based proteomics platforms for high-throughput screening

  • Combination with single-cell RNA sequencing to correlate protein and transcript levels

• Advanced Imaging Applications:

  • Super-resolution microscopy to visualize TM4SF20 distribution within membrane microdomains

  • Expansion microscopy to achieve nanoscale resolution of TM4SF20 localization

  • Live-cell imaging using secondary detection systems compatible with the biotin conjugate

• Proximity Labeling Approaches:

  • Biotin-based proximity labeling to identify proteins interacting with TM4SF20 in native cellular contexts

  • Integration with BioID or APEX2 technologies for spatial proteomics

• Therapeutic Development Applications:

  • Screening for compounds that modulate TM4SF20 expression or function

  • Development of targeted delivery systems for potential therapeutic interventions

  • Biomarker development for TM4SF20-associated disorders

• Extracellular Vesicle (EV) Analysis:

  • Detection of TM4SF20 in EVs as potential biomarkers

  • Investigation of TM4SF20's role in EV biogenesis or cargo selection

These emerging applications would benefit from the antibody's high specificity and its biotin conjugation, which provides flexibility for integration with various detection systems while maintaining sensitivity .

What quality control parameters should researchers verify before using this antibody in critical experiments?

Before employing the Biotin-conjugated TM4SF20 antibody in critical experiments, researchers should verify several key quality control parameters:

• Certificate of Analysis Verification:

  • Confirm lot-specific information including:

    • Protein concentration (typically >95% purity)

    • Biotin-to-antibody ratio

    • Production date and expiration date

    • Species reactivity confirmation (human TM4SF20)

• Functional Validation:

  • Perform pilot experiments with positive control samples

  • Verify signal specificity using appropriate negative controls

  • Confirm detection sensitivity matches manufacturer specifications (detection range 0.32-20 ng/mL for ELISA applications)

  • Assess batch-to-batch reproducibility if using multiple lots

• Physical Inspection:

  • Check for signs of degradation (precipitation, cloudiness)

  • Verify proper storage conditions were maintained during shipping

  • Ensure proper reconstitution if lyophilized

• Application-Specific Validation:

  • Confirm linearity of detection across relevant concentration ranges

  • Verify matrix compatibility with experimental samples

  • Assess potential interference from sample components

• Documentation Requirements:

  • Maintain detailed records of antibody lot information

  • Document all validation experiments

  • Consider including antibody validation data in research publications

Implementing these quality control measures helps ensure experimental reproducibility and reliable data interpretation .

How should researchers analyze and interpret quantitative data generated using this antibody in ELISA applications?

Proper analysis and interpretation of quantitative data from TM4SF20 ELISA applications require rigorous methodological approaches:

• Standard Curve Analysis:

  • Employ four-parameter logistic (4-PL) curve fitting as recommended by manufacturers

  • Alternatively, construct a standard curve on a log/log graph by plotting mean absorbance versus concentration

  • Ensure adequate R² values (typically >0.98) for reliable quantification

  • Verify that samples fall within the linear range of the standard curve

• Data Processing Steps:

  • Subtract average zero standard optical density from all readings

  • Calculate mean values for duplicate/triplicate measurements

  • Apply dilution factor corrections for samples that required dilution

  • Consider applying curve linearization by plotting log(concentration) versus log(OD)

• Statistical Considerations:

  • Account for both intra-assay (CV<10%) and inter-assay (CV<15%) variation

  • Implement appropriate statistical tests based on experimental design

  • Consider power analysis for adequate sample sizing

  • Address outliers according to pre-established criteria

• Result Interpretation:

  • Compare results against appropriate reference ranges

  • Consider biological context when interpreting TM4SF20 levels

  • Account for recovery rates in different matrices (serum: 88%, cell culture media: 84%)

  • Interpret results in relation to controls and experimental manipulations

• Reporting Standards:

  • Document all analysis parameters and statistical approaches

  • Report both absolute values and normalized data when appropriate

  • Include measures of variability (standard deviation, standard error)

  • Clearly state detection limits and dynamic range

This methodical approach to data analysis ensures robust quantitative interpretations of TM4SF20 levels across experimental conditions .

What are the recommended approaches for troubleshooting non-specific binding or high background issues?

When encountering non-specific binding or high background issues with the Biotin-conjugated TM4SF20 antibody, researchers should implement systematic troubleshooting strategies:

• Antibody Dilution Optimization:

  • Perform systematic titration experiments to identify optimal concentration

  • Test dilutions ranging from 1:100 to 1:10,000

  • Balance specific signal strength against background levels

• Blocking Protocol Enhancement:

  • Extend blocking time (1-2 hours at room temperature or overnight at 4°C)

  • Evaluate alternative blocking agents (BSA, casein, non-fat dry milk)

  • Consider addition of non-specific IgG from the same host species as samples

• Wash Procedure Modification:

  • Increase number of wash steps (5-7 washes rather than standard 3)

  • Extend wash duration (1-2 minutes per wash)

  • Add detergent (0.05-0.1% Tween-20) to wash buffer

  • Ensure complete removal of wash buffer between steps

• Sample Preparation Refinement:

  • Pre-clear samples with Protein G beads to remove potentially interfering components

  • Implement additional centrifugation steps to eliminate particulates

  • Consider sample dilution to reduce matrix effects

  • Pre-absorb samples with non-specific proteins or beads

• Detection System Evaluation:

  • Assess potential streptavidin binding to endogenous biotin in samples

  • Test alternative streptavidin conjugates (HRP vs. fluorescent)

  • Implement additional blocking of endogenous biotin if necessary

  • Consider biotin blocking kits for samples with high endogenous biotin

• Reagent Quality Assessment:

  • Prepare fresh working solutions of all reagents

  • Filter buffers to remove particulates

  • Verify proper storage conditions for all components

  • Consider testing a different lot of antibody if available