TPSB2 Recombinant Monoclonal Antibody

What is TPSB2 and what is its biological significance?

TPSB2 is a protein-coding gene belonging to the trypsin-like serine proteases family (peptidase family S1). Tryptases are enzymatically active only as heparin-stabilized tetramers and exhibit remarkable resistance to all known endogenous proteinase inhibitors. The TPSB2 gene is located on chromosome 16p13.3 as part of a tryptase gene cluster with several distinctive features, including a highly conserved 3' UTR and tandem repeat sequences at both the 5' flank and 3' UTR that likely regulate mRNA stability . TPSB2 also contains an unusual intron immediately upstream of the initiator Met codon, separating the transcription initiation site from the protein coding sequence - a characteristic feature of tryptases but uncommon in other genes .

Beta tryptases, including TPSB2, constitute the primary isoenzymes expressed in mast cells, while alpha-tryptases predominate in basophils. These enzymes have been implicated as mediators in the pathogenesis of asthma and various allergic and inflammatory disorders . Understanding TPSB2 function is crucial for investigating mast cell activation pathways and developing therapeutic strategies for allergic conditions.

How do recombinant monoclonal antibodies against TPSB2 differ from conventional antibodies?

Recombinant monoclonal antibodies to TPSB2 offer several advantages over traditional hybridoma-derived antibodies:

Recombinant antibodies like those described in the search results are produced using protein engineering techniques that ensure higher consistency and defined characteristics. For instance, the recombinant human monoclonal antibody to TPSB2 (A324275) exhibits >90% purity as determined by SDS-PAGE and SEC-HPLC, with carefully controlled formulation free of preservatives like sodium azide . This makes it particularly suitable for sensitive applications like functional assays and in vivo studies where contaminants could interfere with results.

What research applications are TPSB2 antibodies validated for?

TPSB2 antibodies have been validated for multiple research applications, enabling comprehensive investigation of mast cell biology:

• Western Blot (WB): Typically used at dilutions of 1:500-1:2000, allowing detection of TPSB2 protein at its predicted molecular weight of approximately 31kDa .

• Immunohistochemistry (IHC-P): Validated for paraffin-embedded sections at dilutions ranging from 1:50-1:1000, enabling visualization of mast cells in tissue samples .

• Enzyme-Linked Immunosorbent Assay (ELISA): Recommended starting concentration of 1 μg/mL, though optimization is required for specific assay conditions .

• Flow Cytometry (FACS): Enables detection and quantification of TPSB2-expressing cells .

• Functional Assays: Certain antibodies like the recombinant human monoclonal (A324275) are specifically validated for functional studies exploring tryptase activity .

• In Vivo Studies: Select antibodies with low endotoxin content and azide-free formulations are suitable for in vivo applications .

• Immunoprecipitation (IP): Some antibodies have been validated for IP at dilutions of approximately 1:50 .

What criteria should researchers consider when selecting a TPSB2 antibody?

Selecting the appropriate TPSB2 antibody requires careful consideration of several factors:

• Experimental Application: Different applications require antibodies with specific characteristics. For instance:

  • For WB, select antibodies validated at the appropriate dilution range (1:500-1:2000)

  • For IHC-P, consider antibodies with demonstrated tissue reactivity and appropriate antigen retrieval protocols

  • For in vivo studies, select antibodies with low endotoxin content and azide-free formulations

• Species Reactivity: Confirm cross-reactivity with your experimental model. Many TPSB2 antibodies react with human, mouse, and rat TPSB2, but specificity should be verified .

• Clonality and Host: Options include:

  • Recombinant human monoclonal (IgG4SP isotype)

  • Rabbit monoclonal (various clones including ARC2328, FHH-20)

  • Rat monoclonal (clone 286820)

• Epitope Recognition: Consider which region of TPSB2 the antibody targets. For example, some antibodies target amino acids 30-275 of human TPSB2 .

• Formulation: Available as:

  • Liquid formulations

  • Lyophilized preparations requiring reconstitution (typically at 0.5-1 mg/mL in sterile PBS)

• Storage Requirements: Most require storage at -20°C to -80°C, with reconstituted antibodies often needing aliquoting to avoid freeze/thaw cycles .

How can researchers validate the specificity of TPSB2 antibodies?

Validating antibody specificity is crucial for reliable results. A comprehensive validation strategy should include:

• Cross-reactivity Testing: Verify the absence of cross-reactivity with related proteins. For example, certain TPSB2 antibodies show no cross-reactivity with recombinant human TPS1, TPSG1, recombinant mouse TPS5, MCPT1, MCPT6, MCPT7, or MCPT11 in direct ELISAs and Western blots .

• Positive and Negative Controls:

  • Positive controls: Mouse skin and rat skin tissues are effective positive controls for IHC applications

  • Negative controls: Tissues known not to express TPSB2 or samples from knockout models

• Blocking Peptide Experiments: Perform experiments with the immunizing peptide to confirm specificity. Some manufacturers offer blocking peptides derived from the human TPSB2 sequence used as immunogen .

• Multiple Application Validation: Confirm antibody performance across different techniques (WB, IHC, ELISA) to ensure consistent target recognition across different protein conformations.

• Molecular Weight Verification: Confirm detection at the expected molecular weight (calculated MW: 31kDa), while noting that observed MW may vary (up to 100kDa in some tests) due to post-translational modifications .

What is the proper procedure for reconstituting lyophilized TPSB2 antibodies?

Proper reconstitution is critical for maintaining antibody activity:

• Reconstitution Solution: Most TPSB2 antibodies should be reconstituted in sterile PBS. For example:

  • Thermo Fisher's TPSB2 monoclonal antibody: Reconstitute at 0.5 mg/mL in sterile PBS

  • Recombinant human monoclonal (A324275): Reconstitute with 100μl of sterile double-distilled water to bring antibody to 1mg/ml concentration

• Reconstitution Method:

  • Allow the lyophilized antibody to reach room temperature before opening

  • Add the recommended volume of reconstitution buffer

  • Gently rotate or swirl to dissolve completely

  • Do not vortex, as this can cause protein denaturation

• Post-Reconstitution Handling:

  • For the recombinant human monoclonal antibody (A324275), after reconstitution, aliquot and store at -80°C

  • Avoid repeated freeze-thaw cycles

  • The reconstituted antibody is stable for approximately one year when properly stored

For Western Blot:

• Tissue/Cell Lysis: Use appropriate lysis buffers containing protease inhibitors to prevent degradation

• Protein Quantification: Standardize protein amounts (typically 25μg per lane)

• Sample Denaturation: Heat samples in reducing sample buffer

• Gel Selection: Use 10-12% SDS-PAGE gels for optimal resolution of the 31kDa TPSB2 protein

• Transfer and Blocking: After transfer, block with 3% nonfat dry milk in TBST

• Primary Antibody Incubation: Use at dilutions of 1:500-1:2000

• Detection: HRP-conjugated secondary antibody with ECL detection system

For Immunohistochemistry:

• Fixation: Formalin fixation and paraffin embedding

• Antigen Retrieval: High pressure antigen retrieval with 0.01M Tris-EDTA Buffer (pH 9.0) prior to IHC staining

• Section Thickness: 4-6μm sections are recommended

• Blocking: Block endogenous peroxidase and non-specific binding

• Primary Antibody: Apply at dilutions ranging from 1:50-1:1000 (can be as high as 1:40000 for some antibodies)

• Incubation: Typically overnight at 4°C or 1-2 hours at room temperature

• Detection: Appropriate HRP-conjugated secondary antibody and DAB substrate

What controls should be included in experimental designs using TPSB2 antibodies?

A robust experimental design requires proper controls:

• Positive Controls:

  • Tissues known to express high levels of TPSB2 (e.g., mouse skin, rat skin, human small intestine)

  • Cell lines with confirmed TPSB2 expression

• Negative Controls:

  • Tissues/cells known not to express TPSB2

  • Primary antibody omission control

  • Isotype control (matching IgG class from the same species)

• Technical Controls:

  • For WB: Molecular weight markers to confirm protein size

  • For IHC: Serial dilution of primary antibody to determine optimal concentration

  • For ELISA: Standard curve with recombinant TPSB2 protein

• Validation Controls:

  • Blocking peptide competition (if available)

  • siRNA knockdown samples

  • CRISPR/Cas9 knockout samples (if available)

How can TPSB2 antibodies be utilized to study mast cell activation in inflammatory disorders?

TPSB2 antibodies are valuable tools for investigating mast cell involvement in inflammatory conditions:

• Tissue Distribution Analysis: Using IHC to map mast cell distribution in healthy versus diseased tissues. Human small intestine sections have been successfully used to demonstrate TPSB2 expression patterns in mast cells .

• Mast Cell Activation Studies: Measuring tryptase release as a marker of mast cell degranulation in response to various stimuli. The recombinant human monoclonal antibody (A324275) is validated for functional assays that can detect tryptase activity following mast cell activation .

• Comparative Analysis: Beta tryptases (including TPSB2) are the main isoenzymes in mast cells, while alpha-tryptases predominate in basophils . This distinction allows researchers to differentiate between mast cell-specific and basophil-specific responses in allergic reactions.

• Disease Mechanism Investigation: TPSB2 antibodies help elucidate the role of mast cell tryptase in asthma and other allergic conditions . By quantifying TPSB2 levels in bronchoalveolar lavage fluid or tissue biopsies, researchers can correlate tryptase activity with disease severity.

• Therapeutic Target Validation: In vivo studies using low-endotoxin, azide-free antibody preparations can help validate TPSB2 as a therapeutic target .

What methods can be employed to differentiate between beta II and beta III tryptase isoforms?

The alleles of TPSB2 show unusual sequence variation, originally thought to represent two separate genes (beta II and beta III) . Differentiating between these isoforms requires specialized approaches:

• Sequence-Specific Antibodies: While most available antibodies recognize both beta II and beta III tryptases, sequence-specific antibodies targeting unique epitopes can be developed for isoform discrimination.

• Mass Spectrometry Analysis: Liquid chromatography-tandem mass spectrometry (LC-MS/MS) can identify isoform-specific peptides after tryptic digestion of immunoprecipitated samples.

• PCR-Based Methods: RT-PCR with isoform-specific primers can quantify mRNA expression of specific beta tryptase variants.

• Computational Analysis: Structural analysis using homology modeling based on the known sequences of beta II and beta III can identify potential epitopes for selective antibody development.

• Functional Enzymatic Assays: Subtle differences in substrate specificity between isoforms can be exploited using tailored enzymatic assays combined with blocking antibodies.

How can researchers optimize TPSB2 antibody use in multiplex immunofluorescence studies?

Multiplex immunofluorescence enables simultaneous detection of multiple markers alongside TPSB2:

• Antibody Panel Design:

  • Select TPSB2 antibodies compatible with multiplexing (consider species, isotype)

  • Combine with markers for mast cell subtypes (e.g., chymase, CD117/c-kit)

  • Include inflammatory markers relevant to the research question

• Sequential Staining Protocol:

  • Begin with the most sensitive antibody (often TPSB2)

  • Use tyramide signal amplification for weak signals

  • Include proper spectral unmixing controls

• Cross-Reactivity Mitigation:

  • Test each antibody individually before multiplexing

  • Use monoclonal antibodies where possible to reduce cross-reactivity

  • Employ appropriate blocking between sequential staining steps

• Data Analysis Strategies:

  • Quantify co-localization of TPSB2 with other markers

  • Analyze spatial relationships between TPSB2+ cells and other cell types

  • Employ machine learning algorithms for unbiased cell classification

Why might TPSB2 appear at different molecular weights in Western blot analysis?

The discrepancy between calculated (31kDa) and observed molecular weights (which can appear as high as 100kDa) can be attributed to several factors:

• Post-translational Modifications: Glycosylation, phosphorylation, and other modifications can significantly alter protein migration on SDS-PAGE.

• Tetramer Formation: Tryptases are enzymatically active as heparin-stabilized tetramers . Incomplete denaturation can result in detection of tetrameric forms (~120-130kDa).

• Sample Preparation Conditions: Variations in reducing conditions, buffer composition, or heating time can affect protein conformation and SDS binding.

• Protein-Protein Interactions: Strong interactions with other proteins that persist during sample preparation can cause shifts in apparent molecular weight.

• Technical Factors: Gel percentage, running conditions, and transfer efficiency can all influence apparent molecular weight.

To address these issues:

• Use fresh sample preparation with stringent denaturing conditions

• Include molecular weight markers

• Validate with alternative methods (e.g., mass spectrometry)

• Consider using multiple antibodies targeting different epitopes

What strategies can minimize background signal in TPSB2 immunohistochemistry?

High background is a common challenge in TPSB2 immunohistochemistry that can be addressed through several approaches:

• Optimize Antibody Dilution: Titrate the primary antibody to determine the optimal concentration. Some TPSB2 antibodies perform well at high dilutions (1:40000) , which can significantly reduce background.

• Enhance Blocking Procedures:

  • Use species-appropriate serum (5-10%) for blocking

  • Include protein blockers like BSA (1-3%)

  • Consider commercial blocking reagents specifically designed to reduce background

• Refine Antigen Retrieval:

  • Optimize antigen retrieval conditions (high-pressure antigen retrieval with 0.01M Tris-EDTA Buffer, pH 9.0 has proven effective)

  • Test different retrieval buffers (citrate vs. EDTA)

  • Adjust retrieval duration and temperature

• Modify Washing Procedures:

  • Increase washing duration and frequency

  • Use detergent-containing wash buffers (0.1% Tween-20 in PBS)

  • Consider gentle agitation during washing steps

• Reduce Endogenous Enzyme Activity:

  • Block endogenous peroxidase (3% H₂O₂ in methanol for 10-15 minutes)

  • For fluorescence applications, include autofluorescence quenching steps

• Antibody Quality Control:

  • Use high-quality, validated antibodies with proven specificity

  • Consider recombinant monoclonal antibodies with enhanced specificity

  • Store antibodies according to manufacturer recommendations to prevent degradation

How should researchers interpret conflicting results between different TPSB2 detection methods?

Discrepancies between different detection methods require systematic investigation:

• Method-Specific Limitations:

  • WB detects denatured proteins while IHC and ELISA detect proteins in more native conformations

  • Different epitopes may be accessible in different methods

  • Some antibodies perform better in specific applications

• Cross-Validation Approach:

  • Employ multiple antibodies targeting different epitopes

  • Use complementary techniques (e.g., mRNA detection via RT-PCR)

  • Include positive and negative controls for each method

• Troubleshooting Checklist:

  • Verify antibody specificity in each application

  • Check sample preparation protocols for compatibility with each method

  • Ensure proper controls are included for each technique

• Data Integration Framework:

  • Weigh results based on technical robustness of each method

  • Consider biological context and expected expression patterns

  • Develop a consensus interpretation that accounts for methodological differences

• Experimental Design Refinement:

  • Modify protocols to standardize conditions across methods where possible

  • Design follow-up experiments to resolve discrepancies

  • Consider advanced techniques (e.g., mass spectrometry) to resolve conflicting results