44 kDa cell wall Antibody

What are the common 44 kDa antigens targeted by antibodies in immunological research?

Several significant 44 kDa proteins serve as important antigens in research contexts. The major outer membrane proteins (OMPs) of the human granulocytic ehrlichiosis (HGE) agent, with molecular sizes of 44-47 kDa, are immunodominant antigens in human infection . CD336 (NKp44) is a 44-kDa transmembrane glycoprotein belonging to the natural cytotoxicity receptor (NCR) family expressed on activated NK cells . Additionally, the 44-kDa processed form of Membrane Type 1 Matrix Metalloproteinase (MT1-MMP) represents another significant 44 kDa protein studied in cancer research contexts . Each of these represents distinct research areas where 44 kDa antibodies are critical investigational tools.

How do I determine the specificity of a 44 kDa antibody against my target protein?

Determining antibody specificity requires multiple validation approaches. Western blot analysis is essential - for example, monoclonal antibodies against HGE agent recognized specific 44-47 kDa proteins in the target isolates but not in unrelated bacteria or host cells . Complementary approaches should include immunofluorescent-antibody staining and immunogold labeling, which can confirm that the recognized antigens are present on the appropriate cellular structures (such as bacterial membranes) . For recombinant proteins, validation should include testing antibody recognition of the recombinant form alongside the native protein, as demonstrated with MAbs 5C11 and 5D13 which recognized both native and recombinant 44-kDa protein, while MAb 3E65 recognized only the native form .

What are the optimal methods for screening hybridoma clones producing antibodies against 44 kDa targets?

Effective screening of hybridoma clones requires a multi-tiered approach. Initially, employ both immunofluorescence assay (IFA) and enzyme-linked immunosorbent assay (ELISA) in parallel. For IFA screening, apply undiluted supernatant to target antigen slides, incubate at 37°C for 90 minutes, then wash with 2× PBS containing 0.05% Tween 20 . Follow with fluorescein isothiocyanate-conjugated anti-mouse immunoglobulin detection antibodies (1:100 dilution, 90 minutes at 37°C) . For ELISA screening, coat plates with purified target antigen, add hybridoma supernatants, then detect with enzyme-conjugated secondary antibodies . Establish clear cutoff values (e.g., absorbance >0.8 at 405nm) and confirm positive clones with both methods . Finally, subclone positive hybridomas three times via limiting dilution technique to ensure monoclonality and stability .

What is the optimal procedure for producing monoclonal antibodies against 44 kDa membrane proteins?

Production of high-quality monoclonal antibodies against 44 kDa membrane proteins requires careful immunization and hybridoma generation. Begin by immunizing BALB/c mice with purified target antigen in appropriate adjuvant until antibody titers exceed 1:2,560 . Harvest spleen cells and fuse with myeloma cells using either 50% polyethylene glycol 4000 or 40% polyethylene glycol 4000 containing 7.6% dimethyl sulfoxide in PBS . Culture the fusion mixture in selective medium containing hypoxanthine, aminopterin, and thymidine (HAT) to eliminate unfused myeloma cells . Screen for specific antibody production using both IFA and ELISA techniques as described earlier. For membrane proteins like the 44 kDa OMP of HGE agent, ensure purification maintains native conformation to generate antibodies that recognize structural epitopes . Subclone positive hybridomas three times via limiting dilution to ensure monoclonality before antibody production scale-up .

How should I standardize flow cytometric analysis when using antibodies against 44 kDa cell surface markers?

For standardized flow cytometric analysis with antibodies against 44 kDa cell surface markers, precise titration is critical. Determine optimal antibody concentration empirically, starting with approximately 0.06-0.25 μg per test (where a test represents antibody amount in a 100 μL sample volume) . Cell numbers can range from 10^5 to 10^8 cells per test, but should be standardized across experiments . For fluorophore selection, consider the spectral characteristics of your target - for example, Alexa Fluor 700-conjugated antibodies emit at 723 nm and require red laser (633 nm) excitation with appropriate filter sets (685 LP mirror and 710/20 filter) . Always include appropriate isotype controls and compensation controls if using multiple fluorophores. For quantitative comparison between samples, establish consistent gating strategies and ensure all parameters for brightness and contrast remain constant throughout the experiment . Calculate and report the average intensity of fluorescence signal per cell across multiple independent experiments (n≥3) with appropriate statistical analysis .

What purification techniques yield the highest quality 44 kDa antibodies for research applications?

High-quality antibody purification for research applications demands rigorous quality control standards. After hybridoma culture, purify antibodies using affinity chromatography (typically protein A/G for IgG isotypes) followed by size-exclusion chromatography to remove aggregates . Filter the final product through 0.2 μm filters to ensure sterility . Evaluate purity by SDS-PAGE (aim for >90% purity) , and assess aggregation levels using high-performance liquid chromatography (HPLC), targeting <10% aggregation . For therapeutic or critical research applications, test endotoxin levels using Limulus Amebocyte Lysate (LAL) assay, aiming for <0.001 ng/μg antibody . For functional applications, validate activity through appropriate bioassays specific to your research context. When working with antibodies against membrane proteins like 44 kDa outer membrane proteins, ensure proper folding and epitope exposure are maintained throughout purification to preserve recognition of conformational epitopes .

How do 44 kDa cell surface proteins regulate cellular functions in different biological systems?

The 44 kDa cell surface proteins exhibit diverse functional roles across biological systems. CD44, a transmembrane glycoprotein expressed on hematopoietic and non-hematopoietic cells, functions as an adhesion molecule by binding to hyaluronate, an extracellular matrix component . This interaction mediates critical processes including cell-cell adhesion, migration, lymphocyte activation, recirculation, and homing . In contrast, CD336 (NKp44), a 44-kDa transmembrane glycoprotein on activated NK cells, requires association with DAP12 (an ITAM-containing transmembrane accessory protein) for expression and function . CD336 participates in innate immune responses against viral infections and tumor cells . In bacterial systems, the 44-47 kDa outer membrane proteins of the HGE agent serve as immunodominant antigens during human infection . Meanwhile, the 44-kDa processed form of MT1-MMP, though lacking the catalytic domain, paradoxically enhances enzymatic activity by delaying the endocytosis of active MT1-MMP, resulting in higher levels of active enzyme at the cell surface . These examples illustrate how 44 kDa proteins regulate diverse cellular functions through different mechanistic pathways.

What is the mechanistic relationship between active enzymes and their 44 kDa processed forms?

The relationship between active enzymes and their 44 kDa processed forms reveals complex regulatory mechanisms. Taking MT1-MMP as a model system, autocatalytic processing generates an inactive membrane-tethered 44-kDa product (44-MT1) lacking the catalytic domain . Contrary to expected inhibitory effects of losing the catalytic domain, this processed form enhances enzymatic functions through several mechanisms. Expression of recombinant 44-MT1 in HT1080 fibrosarcoma cells results in enhanced pro-MMP-2 activation, increased proliferation within three-dimensional collagen I matrices, and accelerated tumor growth and metastasis . Mechanistically, the 44-kDa form delays the rate of active MT1-MMP endocytosis, maintaining higher levels of active enzyme at the cell surface . This effect depends on the cytosolic domain, as its deletion eliminates the stimulatory effects . Additionally, the hinge region plays a critical role in this functional relationship - deletion of the hinge converts 44-MT1 from a positive to a negative regulator of enzyme function both in vitro and in vivo . This complex interplay between processed and active forms represents an important regulatory mechanism controlling enzyme availability and function at the cell surface.

How do monoclonal antibodies against 44 kDa proteins facilitate understanding of epitope heterogeneity?

Monoclonal antibodies against 44 kDa proteins serve as powerful tools for dissecting epitope heterogeneity across isolates and related proteins. In studies of the HGE agent, three monoclonal antibodies (3E65, 5C11, and 5D13) revealed distinct patterns of epitope conservation and variability across isolates . While MAbs 5C11 and 5D13 recognized conserved epitopes across all six isolates examined, MAb 3E65 displayed specificity for only the homologous human isolate, recognizing a 44-kDa protein absent in the other five isolates . This selective recognition pattern demonstrated the presence of both conserved and variable epitopes within the 44-47 kDa protein family . Further epitope characterization revealed that MAbs 5C11 and 5D13 recognized epitopes present on both native and recombinant 44-kDa protein, while MAb 3E65 recognized only native conformational epitopes lost in the recombinant form . These findings enabled researchers to map the molecular topology of the protein and understand strain-specific variations. Similar approaches can be applied to other 44 kDa proteins like CD44, where alternative splicing generates multiple isoforms with distinct functional domains that can be distinguished using carefully selected monoclonal antibodies .

What are the experimental considerations when using 44 kDa protein antibodies for in vivo passive immunization studies?

Passive immunization studies with antibodies against 44 kDa proteins require careful experimental design. When evaluating protective efficacy, compare multiple antibody clones targeting different epitopes of the same protein. For instance, passive immunization with MAb 3E65 (targeting a specific 44-kDa protein epitope) provided greater protection against HGE agent infection in mice compared to MAbs 5C11 and 5D13 (targeting different epitopes) . This differential protection highlights the importance of epitope selection in determining protective efficacy. Prior to in vivo studies, characterize antibody properties including isotype, binding affinity, epitope specificity, and functional activity in relevant in vitro assays . For dosing, begin with established protocols in the literature, typically 100-500 μg of antibody per mouse depending on body weight, and consider pharmacokinetic properties of the specific antibody . Include appropriate controls including isotype-matched irrelevant antibodies and untreated groups. When reporting results, document protection metrics including pathogen burden, survival rates, and immunological parameters, with statistical analysis comparing all experimental groups . Additionally, collect serum throughout the experiment to monitor antibody persistence and potential anti-antibody responses.

How can quantitative fluorescence-based antibody uptake assays be optimized for studying 44 kDa membrane protein trafficking?

Optimizing quantitative fluorescence-based antibody uptake assays for 44 kDa membrane protein trafficking requires precise methodological controls. Begin by labeling cells with fluorophore-conjugated antibodies against your 44 kDa target protein at 4°C (which permits binding but prevents endocytosis) . After removing unbound antibody, warm cells to 37°C to initiate internalization, and collect samples at defined time points . For image acquisition, use consistent parameters - excite fluorophores with appropriate lasers (e.g., 488 nm argon laser) and maintain identical brightness and contrast settings across all samples and time points . Identify cells using nuclear counterstains such as DAPI, and measure average fluorescence intensity per cell using image analysis software like Metamorph . Always perform experiments in triplicate and report means with standard deviations . For mechanistic studies of trafficking modulators, include positive and negative controls that affect endocytosis rates. This approach allowed researchers to demonstrate that expression of the 44-kDa form of MT1-MMP delays the endocytosis of active MT1-MMP, resulting in higher levels of active enzyme at the cell surface - a finding with significant implications for understanding the regulation of membrane proteases .

How do I troubleshoot antibody cross-reactivity with other proteins in the 44 kDa range?

Cross-reactivity troubleshooting for 44 kDa antibodies requires systematic control experiments. First, perform Western blot analysis using both your target sample and known negative controls . For instance, when characterizing antibodies against the 44 kDa OMP of HGE agent, researchers tested reactivity against five human HGE isolates, one tick isolate, and negative controls . Second, evaluate recognition patterns - antibodies may recognize homologous proteins across related isolates but with different molecular weight patterns (e.g., 44-47 kDa range for HGE OMP antibodies) . Third, use purified protein fractions - test antibody reactivity against isolated outer membrane protein fractions to confirm specificity to membrane components rather than cytosolic proteins . Fourth, employ recombinant protein validation - compare antibody recognition of recombinant versus native proteins, as some antibodies (like MAb 3E65) may recognize only native conformational epitopes . Fifth, use complementary detection methods including immunofluorescence and immunogold labeling to confirm proper subcellular localization of the recognized antigen . Finally, consider epitope mapping or competitive binding assays with known ligands to further characterize specificity. When cross-reactivity cannot be eliminated, document the cross-reactive species and their expression patterns to facilitate accurate data interpretation.

What controls are essential when studying the effects of 44 kDa protein processing on cellular function?

When investigating how 44 kDa protein processing affects cellular function, implement comprehensive controls to distinguish direct effects from artifacts. First, include full-length protein expression controls alongside the processed 44 kDa form to directly compare functional outcomes . Second, create domain deletion variants - for processed forms like the 44-kDa MT1-MMP fragment, systematically delete individual domains (cytosolic domain, hinge region) to identify which regions mediate the observed effects . Third, utilize catalytically inactive mutants of the full-length protein to distinguish effects dependent on enzymatic activity versus protein-protein interactions . Fourth, employ multiple functional readouts spanning different biological levels - for MT1-MMP, researchers measured pro-MMP-2 activation (biochemical), proliferation in collagen matrices (cellular), and tumor growth and metastasis (organismal) . Fifth, conduct experiments across multiple cell types to ensure the observed effects are not cell-line specific . Sixth, perform time-course analyses to distinguish immediate versus delayed consequences of protein processing. Finally, implement both gain-of-function (expressing the 44 kDa form) and loss-of-function (preventing processing) approaches to comprehensively characterize the role of processing in your system .

How can I distinguish between direct effects of antibodies on 44 kDa proteins versus secondary effects on associated signaling complexes?

Distinguishing direct versus secondary antibody effects requires mechanistic dissection of protein complexes. First, characterize the protein interaction network of your 44 kDa target through co-immunoprecipitation or proximity labeling techniques . For instance, CD336 (NKp44) requires association with DAP12 for function, creating potential for indirect effects . Second, use domain-specific antibodies targeting distinct regions of the 44 kDa protein to identify which interactions are disrupted by antibody binding . Third, compare effects of whole antibodies versus Fab fragments - whole antibodies can crosslink receptors and trigger signaling, while Fab fragments generally cannot . This is particularly relevant for receptor crosslinking studies like those with the 44.189 antibody against CD336 . Fourth, implement time-course studies to distinguish immediate (likely direct) versus delayed (potentially secondary) effects. Fifth, conduct parallel experiments with known signaling inhibitors targeting pathways downstream of your 44 kDa protein. Sixth, utilize cells expressing mutant forms of the 44 kDa protein that cannot engage specific interaction partners. Finally, complement antibody studies with genetic approaches (siRNA, CRISPR) targeting the 44 kDa protein or its interaction partners to confirm mechanism attribution .