THNSL2 Antibody, Biotin conjugated

What is THNSL2 and what is its biological function?

THNSL2 (threonine synthase-like 2, also known as TSH2) is a 484 amino acid protein belonging to the threonine synthase family. It functions as a catabolic phospholyase on both gamma- and beta-phosphorylated substrates. Using pyridoxal phosphate as a cofactor, THNSL2 degrades O-phospho-threonine (PThr) to alpha-ketobutyrate, ammonia, and phosphate. The protein exists in four alternatively spliced isoforms and is encoded by a gene mapping to human chromosome 2p11.2 . THNSL2 is also known as Secreted osteoclastogenic factor of activated T-cells (SOFAT) in some research contexts .

What are the key characteristics of THNSL2 Antibody, Biotin conjugated?

THNSL2 Antibody, Biotin conjugated (targeting AA 295-441) is a rabbit polyclonal antibody with the following specifications:

• Host: Rabbit

• Clonality: Polyclonal

• Isotype: IgG

• Purification: Protein G purified with >95% purity (by SDS-PAGE)

• Conjugate: Biotin

• Immunogen: Recombinant Human Threonine synthase-like 2 protein (295-441AA)

• Primary application: ELISA

• Reactivity: Human

What are the common applications for THNSL2 Antibody, Biotin conjugated?

The biotin-conjugated THNSL2 antibody is primarily designed for ELISA applications, though the unconjugated versions of similar antibodies have broader application ranges including:

• Western Blotting (WB)

• Immunohistochemistry (IHC)

• Immunofluorescence (IF)

• Flow cytometry/FACS (for some variants)

The biotin conjugation specifically enables applications involving streptavidin detection systems, which can enhance sensitivity and provide opportunities for signal amplification in various experimental contexts .

What species reactivity can be expected with THNSL2 Antibody, Biotin conjugated?

• Some target the same epitope (AA 295-441) but show reactivity with both human and mouse samples

• Others targeting different regions show expanded reactivity including human, mouse, rat, cow, horse, dog, guinea pig, pig, rabbit, and zebrafish with varying degrees of predicted cross-reactivity

This variability highlights the importance of selecting the appropriate antibody based on the specific species being studied in your research.

How does biotin conjugation affect the binding specificity and affinity of THNSL2 antibodies?

Biotin conjugation can impact binding characteristics, though these effects vary depending on conjugation methods and the specific epitope involved. When evaluating THNSL2 antibodies:

• Conjugation chemistry: The biotin molecules are typically attached to lysine residues or other accessible amino groups on the antibody. Since THNSL2 antibodies (like trastuzumab mentioned in comparative studies) contain multiple lysine residues (approximately 41), random biotinylation can potentially occur at both Fab and Fc regions, potentially affecting antigen recognition .

• Comparative binding studies with unconjugated antibodies have shown that biotin-conjugated antibodies may require higher concentrations to reach 50% of the maximal mean fluorescence intensity in binding assays, suggesting some reduction in apparent affinity .

• The molar ratio of biotin to antibody is critical - higher molar ratios (1:6, 1:8) may decrease binding to target antigens compared to lower ratios (1:2, 1:4), as demonstrated in related streptavidin-biotin conjugation studies .

How can streptavidin-biotin systems be leveraged with THNSL2 Antibody for complex experimental designs?

The streptavidin-biotin system offers versatile applications for THNSL2 research through several approaches:

• Signal amplification in detection systems:

  • Biotinylated THNSL2 antibodies can bind multiple streptavidin molecules (each with 4 biotin binding sites), allowing attachment of multiple reporter molecules (enzymes, fluorophores) per antibody

  • This creates significant signal amplification for low-abundance targets

• Targeted delivery systems:

  • Biotinylated THNSL2 antibodies can be conjugated to streptavidin-linked payloads (e.g., Saporin) to create Antibody-Streptavidin-Biotin-Payload complexes

  • This allows rapid evaluation of antibody internalization capacity, critical for applications like targeted toxin delivery

• Multiplexed detection:

  • Different biotinylated antibodies can be used with streptavidin conjugated to distinct fluorophores for multi-target imaging

  • This is particularly valuable for co-localization studies involving THNSL2

These applications can be assembled within approximately 4-7 hours, making this system highly adaptable for screening multiple experimental conditions .

What are the considerations for validating THNSL2 Antibody specificity in diverse experimental contexts?

Comprehensive validation of THNSL2 antibodies requires multiple approaches:

• Western blot validation:

  • Test across multiple cell lines (demonstrated examples: HeLa, HEK-293, HepG2, H9C2, RAW264.7)

  • Expected molecular weight: 45-60 kDa (calculated MW: 45 kDa, observed MW: 55-60 kDa)

  • Include positive and negative control cell lines

• Cross-reactivity assessment:

  • Test against predicted reactive species

  • For THNSL2 Middle Region antibodies, published cross-reactivity data shows:

    Species	Predicted Reactivity
    Human	100%
    Mouse	100%
    Rat	100%
    Horse	100%
    Cow	93%
    Dog	92%
    Guinea Pig	92%
    Pig	93%
    Rabbit	93%
    Zebrafish	80%

• Epitope-specific validation:

  • Compare antibodies targeting different epitopes (AA 295-441 vs. other regions)

  • Validate using recombinant protein fragments or peptide blocking studies

• Application-specific controls:

  • For ELISA: Include recombinant protein standards, isotype controls

  • For IHC/IF: Include peptide competition assays

  • For all applications: Test antibody dilution series to establish optimal working concentrations

What are the recommended protocols for optimizing THNSL2 Antibody, Biotin conjugated in ELISA applications?

For optimal ELISA performance with biotinylated THNSL2 antibody:

• Dilution optimization:

  • Start with manufacturer's recommended dilution (typically 1:500-1:2000)

  • Perform a titration series (1:100, 1:500, 1:1000, 1:2000, 1:5000) to determine optimal signal-to-noise ratio

  • Remember that biotin-conjugated antibodies may require higher concentrations than unconjugated versions

• Detection system optimization:

  • Use high-sensitivity streptavidin-HRP or streptavidin-AP conjugates

  • Compare different streptavidin conjugates for optimal signal development

  • Consider signal amplification systems (e.g., tyramide signal amplification) for low-abundance targets

• Blocking conditions:

  • Use biotin-free blocking reagents to prevent background

  • Optimize blocking time (typically 1-2 hours at room temperature)

  • Consider adding avidin/streptavidin blocking steps if background remains high

• Sample preparation:

  • For cell/tissue lysates: ensure complete protein extraction using appropriate lysis buffers containing protease inhibitors

  • Determine optimal protein concentration for coating (typically 1-10 μg/ml)

  • Establish standard curves using recombinant THNSL2 protein

How should researchers handle and store THNSL2 Antibody, Biotin conjugated to maintain its activity?

Proper handling and storage are crucial for maintaining antibody performance:

• Storage recommendations:

  • Store at -20°C for long-term storage (stable for approximately one year)

  • For short-term/frequent use, store at 4°C for up to one month

  • Avoid repeated freeze-thaw cycles (aliquot upon receipt)

  • Some preparations contain 50% glycerol to minimize freeze-thaw damage

• Buffer composition:

  • Typically supplied in PBS with preservatives like 0.03% Proclin 300 or 0.02% sodium azide

  • Often contains stabilizers like 50% glycerol and/or 0.1% BSA

  • pH is typically maintained at approximately 7.3-7.4

• Handling precautions:

  • Avoid exposure to light (particularly important for biotin conjugates)

  • Work with sterile techniques to prevent microbial contamination

  • Use polypropylene tubes for storage to minimize protein binding to container surfaces

  • When aliquoting, maintain sterile conditions and use appropriate personal protective equipment due to preservatives like sodium azide (hazardous substance)

What troubleshooting approaches can be used when working with THNSL2 Antibody, Biotin conjugated?

When facing challenges with biotinylated THNSL2 antibody experiments:

• For high background issues:

  • Implement avidin/biotin blocking steps to eliminate endogenous biotin

  • Increase washing steps and use more stringent wash buffers (higher salt or detergent concentration)

  • Reduce antibody concentration

  • Use biotin-free blocking reagents

• For weak or no signal:

  • Verify target expression in sample (use positive control cells with known THNSL2 expression)

  • Increase antibody concentration

  • Extend incubation time

  • Check detection system functionality with positive controls

  • Consider antigen retrieval methods for fixed samples

• For inconsistent results:

  • Standardize sample preparation methods

  • Implement rigorous quality control for reagents

  • Validate antibody lot-to-lot consistency

  • Monitor pH and temperature conditions during experiments

  • Test multiple dilutions to establish robustness of signal

• For non-specific binding:

  • Implement additional blocking steps

  • Increase washing stringency

  • Pre-adsorb antibody with proteins from non-target species

  • Use competitive binding with immunizing peptide to confirm specificity

How should researchers interpret varying band sizes when using THNSL2 Antibody in Western blot applications?

When analyzing Western blot results:

• Expected molecular weight patterns:

  • Calculated molecular weight: 45 kDa (405 amino acids)

  • Observed molecular weight: 55-60 kDa in validated cell lysates

  • This discrepancy is common and may reflect post-translational modifications

• Multiple band interpretation:

  • THNSL2 exists in four alternatively spliced isoforms, which may appear as distinct bands

  • Isoform-specific expression varies by tissue/cell type

  • Post-translational modifications (glycosylation, phosphorylation) can alter apparent molecular weight

  • Proteolytic processing may generate fragments with epitope retention

• Validation strategies:

  • Compare band patterns across multiple cell lines (HeLa, HEK-293, HepG2 are validated options)

  • Use recombinant THNSL2 protein as a positive control

  • Consider siRNA knockdown experiments to confirm band specificity

  • Compare results with antibodies targeting different THNSL2 epitopes

What factors might contribute to discrepancies in experimental results between different THNSL2 antibodies?

Several factors can explain variability between different THNSL2 antibodies:

• Epitope differences:

  • THNSL2 antibodies target diverse regions (AA 91-140, 192-204, 232-261, 251-300, 295-441, 811-860)

  • Epitope accessibility varies by application and sample preparation method

  • Some epitopes may be masked by protein interactions or conformational changes

  • Certain epitopes may be absent in specific isoforms

• Technical variations:

  • Different conjugation chemistries (biotin, FITC, HRP) can affect antibody performance

  • Conjugation ratios impact binding efficiency (demonstrated in streptavidin-biotin studies)

  • Clone-to-clone variability in polyclonal antibody preparations

  • Lot-to-lot variations in antibody production

• Application-specific factors:

  • ELISA vs. Western blot vs. IHC applications expose different epitopes

  • Sample preparation methods (denaturing vs. native conditions)

  • Buffer compositions and experimental conditions

  • Detection system sensitivities

• Validation parameters:

  • Different antibodies undergo varying validation processes

  • Some are validated only for specific applications or species

  • Validation standards vary between manufacturers

  • Verification methods (cell lysate vs. recombinant protein) influence performance metrics

How can researchers assess cross-reactivity and non-specific binding when using THNSL2 Antibody, Biotin conjugated?

To evaluate cross-reactivity and ensure specificity:

• Experimental controls:

  • Negative control tissues/cells (known to lack THNSL2 expression)

  • Isotype control antibodies (matched to THNSL2 antibody)

  • Peptide competition assays using immunizing peptide

  • siRNA knockdown of THNSL2 to confirm signal reduction

• Cross-species validation:

  • Compare reactivity across species with predicted homology

  • Review sequence alignment data for epitope conservation

  • Test with recombinant proteins from multiple species

  • Reference predicted reactivity data:

    Species	Predicted Reactivity
    Human	100%
    Mouse	100%
    Rat	100%
    Horse	100%
    Cow	93%
    Dog	92%
    Guinea Pig	92%
    Pig	93%
    Rabbit	93%
    Zebrafish	80%

• Signal validation approaches:

  • Implement titration experiments to assess signal-to-noise ratio

  • Evaluate signal patterns across different experimental conditions

  • Compare results from multiple antibodies targeting different epitopes

  • Analyze consistency across technical and biological replicates

• Streptavidin-biotin specific considerations:

  • Include avidin/biotin blocking steps to eliminate endogenous biotin signals

  • Test for non-specific binding of streptavidin detection reagents

  • Compare biotinylated antibody performance with unconjugated versions

  • Implement appropriate controls for streptavidin-based detection systems