OLFML3 Antibody, Biotin conjugated

What is OLFML3 and what are its key biological functions?

OLFML3 (Olfactomedin-like protein 3) is a secreted scaffold protein belonging to the family of olfactomedin-domain-containing proteins. It functions as a matricellular protein with established proangiogenic properties . At the molecular level, OLFML3 plays several critical biological roles:

• During embryonic development, it is expressed in presumptive vasculogenic regions and serves as an essential regulator of dorsoventral patterning

• It acts as a scaffold protein that facilitates the association between tolloid proteases and their substrate chordin (CHRD), enhancing CHRD degradation

• In adult tissues, OLFML3 expression is primarily limited to tissues undergoing active remodeling

• It interacts with bone morphogenetic protein 4 (BMP4), a proangiogenic factor involved in tumor cell migration and invasion

• Recent research has identified its role in immune regulation, particularly in modulating inflammatory responses during bacterial infection

From a structural perspective, OLFML3 contains a signal peptide, a coiled-coil (CC) domain, and an olfactomedin (OLF) domain, with the OLF domain being crucial for protein-protein interactions .

What are the specifications of commercially available OLFML3 Antibody, Biotin conjugated?

The OLFML3 Antibody, Biotin conjugated is a polyclonal antibody raised in rabbits against recombinant human Olfactomedin-like protein 3 (amino acids 201-406). The antibody specifications include:

This antibody recognizes the human Olfactomedin-like protein 3, also known by its aliases: HNOEL-iso, hOLF44, and OLF44 .

How should OLFML3 antibody be stored and handled to maintain optimal activity?

Proper storage and handling of the OLFML3 antibody is critical to maintain its specificity and activity for experimental applications. Follow these methodological guidelines:

• Upon receipt, aliquot the antibody into smaller volumes to minimize freeze-thaw cycles, which can degrade the antibody and reduce its effectiveness

• Store the aliquots at -20°C for routine use or -80°C for long-term storage

• When removing from storage, thaw aliquots quickly at room temperature and keep on ice while working

• Avoid exposure to light, particularly important for biotin-conjugated antibodies to prevent photobleaching of the conjugate

• The antibody is provided in a stabilizing buffer containing 50% glycerol, 0.01M PBS (pH 7.4), and 0.03% Proclin 300 as a preservative; this formulation helps maintain antibody integrity

• When diluting for applications, use fresh, sterile buffers and prepare only the amount needed for immediate use

• For biotin-conjugated antibodies specifically, avoid buffers containing sodium azide as it can inhibit the activity of horseradish peroxidase if used in detection systems

Following these storage and handling procedures will help ensure consistent experimental results and extend the usable lifetime of the antibody.

How can OLFML3 Antibody, Biotin conjugated be optimized for ELISA applications?

When optimizing OLFML3 Antibody, Biotin conjugated for ELISA applications, several methodological considerations are essential:

• Antibody titration: Perform a checkerboard titration to determine the optimal concentration. While the product information suggests using the antibody at concentrations between 1-5 μg/mL for ELISA, the optimal concentration should be determined experimentally for each specific application .

• Blocking optimization: Use a 3-5% BSA or non-fat milk solution in PBS or TBS to minimize background signal. For biotin-conjugated antibodies specifically, ensure the blocking solution does not contain endogenous biotin that might interfere with detection.

• Detection system selection: Since the antibody is biotin-conjugated, use a streptavidin-conjugated enzyme (HRP or AP) for detection. The high-affinity biotin-streptavidin interaction (Kd ≈ 10^-15) provides excellent sensitivity.

• Incubation parameters:

  • Primary antibody (OLFML3 target capture): 1-2 hours at room temperature or overnight at 4°C

  • OLFML3 Antibody, Biotin conjugated: 1-2 hours at room temperature

  • Streptavidin-enzyme conjugate: 30-60 minutes at room temperature

• Washing optimization: Use PBS-T (PBS + 0.05-0.1% Tween-20) and perform at least 3-5 washing cycles between each step to reduce background.

• Controls to include:

  • Positive control: Recombinant OLFML3 protein

  • Negative control: Omit primary antibody

  • Background control: Omit both primary and secondary antibodies

• Signal development: For HRP-conjugated streptavidin, use TMB substrate and monitor color development, stopping the reaction with H₂SO₄ when appropriate signal-to-noise ratio is achieved.

This methodological approach ensures optimal sensitivity and specificity when using the OLFML3 Antibody, Biotin conjugated in ELISA applications.

Beyond ELISA, what other applications might OLFML3 Antibody, Biotin conjugated be suitable for?

While the product information specifically validates the OLFML3 Antibody, Biotin conjugated for ELISA applications , its biotin conjugation makes it potentially suitable for several other research applications:

• Immunohistochemistry (IHC): The biotin-conjugated antibody can be used with streptavidin-HRP systems for detecting OLFML3 in tissue sections. This would be particularly valuable for studying OLFML3 expression in:

  • Tumor tissues, especially colorectal cancer and lung carcinoma, where OLFML3 promotes vascularization

  • Developing tissues, particularly in presumptive vasculogenic regions

  • Tissues undergoing active remodeling

• Flow cytometry: When paired with fluorescently labeled streptavidin (e.g., streptavidin-PE or streptavidin-APC), the antibody could be used to detect and quantify OLFML3 expression in cell populations.

• Immunoprecipitation: As part of a streptavidin-based pull-down system to isolate OLFML3 and its interacting partners, similar to the proteomics analyses described in the research literature .

• Immunofluorescence microscopy: Using fluorescent streptavidin conjugates for subcellular localization studies, particularly useful for investigating the intracellular vs. extracellular distribution of OLFML3.

• Western blotting: Potentially useful with streptavidin-HRP for detection, especially when analyzing OLFML3 expression in cellular fractions or tissue lysates.

For any application beyond ELISA, researchers should first validate the antibody's performance in their specific experimental system, optimizing parameters such as antibody concentration, incubation times, and detection methods. Based on the literature findings regarding OLFML3's complex role in both extracellular and intracellular compartments, these additional applications could provide valuable insights into OLFML3 biology .

How can OLFML3 Antibody be used to investigate its role in tumor angiogenesis and cancer progression?

OLFML3 antibodies, including biotin-conjugated variants, can be instrumental in investigating OLFML3's role in tumor angiogenesis and cancer progression through several methodological approaches:

• Therapeutic targeting studies: Research has shown that targeting OLFML3 with polyclonal antibodies inhibits tumor growth in mouse models of lung carcinoma . Researchers can design similar studies using biotin-conjugated antibodies to:

  • Evaluate the effect of OLFML3 neutralization on tumor growth kinetics

  • Assess changes in tumor vasculature density and morphology

  • Measure impacts on lymphangiogenesis and pericyte coverage

• Mechanistic investigations: The biotin-conjugated antibody can be used in immunohistochemistry or immunofluorescence to co-localize OLFML3 with markers of:

  • Endothelial cells (e.g., CD31)

  • Pericytes (e.g., NG2, PDGFR-β)

  • Tumor-associated macrophages (TAMs)

  • NKT cells

• Combination therapy evaluation: As indicated in the literature, researchers have investigated whether combining anti-OLFML3 antibodies with anti-PD-1 antibodies produces more efficient antitumor effects than monotherapy . Similar combinatorial approaches could be explored using the biotin-conjugated antibody.

• Expression profiling: Using the antibody in ELISA or immunohistochemistry to:

  • Compare OLFML3 expression across different tumor types

  • Correlate expression levels with clinicopathological features

  • Evaluate OLFML3 as a potential biomarker for tumor angiogenesis

• Protein interaction studies: The biotin tag facilitates pull-down assays to identify protein interaction partners involved in angiogenesis, such as BMP4 .

When designing these experiments, researchers should consider using appropriate controls, including isotype controls and OLFML3 knockout models as described in the literature , to validate antibody specificity and biological effects.

What is known about OLFML3's interaction with IRG1, and how can researchers investigate this relationship?

Recent research has revealed a novel interaction between OLFML3 and IRG1 (Immune-Responsive Gene 1), with significant implications for mitochondrial function and inflammatory responses. Researchers can investigate this relationship using several approaches:

• Domain mapping studies: The literature indicates that OLFML3 interacts with IRG1 via its OLF domain, while IRG1 interacts primarily through its C-terminal α+β domain . To further characterize this interaction:

  • Use truncation constructs of both proteins (as described in the literature) for co-immunoprecipitation experiments

  • The biotin-conjugated OLFML3 antibody can be used to pull down OLFML3-IRG1 complexes if the epitope doesn't interfere with the interaction

• Subcellular localization studies: OLFML3 has been shown to localize to the outer mitochondrial membrane and facilitate IRG1's mitochondrial localization . Researchers can:

  • Use immunofluorescence with the biotin-conjugated OLFML3 antibody (detected with fluorescent streptavidin) alongside mitochondrial markers

  • Perform subcellular fractionation followed by immunoblotting to quantify the distribution of OLFML3 and IRG1 in different cellular compartments

• Functional studies: To investigate the impact of the OLFML3-IRG1 interaction on:

  • Itaconate production (IRG1's metabolic product)

  • Mitochondrial respiratory function

  • Inflammatory responses to LPS or bacterial challenge

• Structural biology approaches: Though not directly utilizing the antibody, researchers might use purified OLFML3 and IRG1 for:

  • Protein crystallography

  • Cryo-EM

  • Hydrogen-deuterium exchange mass spectrometry to map the precise interaction interface

The literature indicates that the intracellular function of OLFML3 (despite its signal peptide) is particularly interesting, as most previous studies focused on its extracellular roles . This presents an opportunity to investigate how the dynamics between intracellular and extracellular OLFML3 are regulated under physiological and pathological conditions.

How can OLFML3 knockout models be effectively utilized alongside OLFML3 antibodies in research?

OLFML3 knockout models provide a powerful complementary approach to antibody-based studies, enabling researchers to validate antibody specificity and investigate OLFML3's biological functions more comprehensively:

• Validation of antibody specificity: OLFML3 knockout tissues or cells serve as critical negative controls for antibody specificity . Researchers should:

  • Compare antibody staining patterns between wild-type and knockout samples

  • Use Western blot analysis to confirm the absence of specific bands in knockout samples

  • Include these controls when establishing new applications for the biotin-conjugated antibody

• Comparative phenotypic analysis: The literature describes OLFML3 knockout mice showing:

  • Reduced survival during LPS-induced sepsis

  • Exacerbated pulmonary edema and inflammatory cell infiltration in acute lung injury models

  • Elevated pro-inflammatory cytokine levels in bronchoalveolar lavage fluid and sera

  These phenotypes can be further characterized using the biotin-conjugated antibody to investigate where and when OLFML3 expression is critical.

• Rescue experiments: To definitively link phenotypes to OLFML3 deficiency:

  • Reintroduce wild-type or mutant OLFML3 into knockout cells/tissues

  • Use the biotin-conjugated antibody to confirm expression

  • Assess restoration of normal function

• Domain-specific functions: Creating knockin mice expressing OLFML3 truncation mutants (e.g., lacking the OLF domain identified as critical for IRG1 interaction ) can help:

  • Dissect the importance of specific protein domains

  • Differentiate between intracellular and extracellular functions

  • The biotin-conjugated antibody can verify expression of these truncated proteins

• Tissue-specific knockout studies: Since global Olfml3 knockout affects survival , generating tissue-specific knockouts using Cre-lox technology may:

  • Avoid developmental lethality

  • Enable study of OLFML3 function in specific organs

  • The biotin-conjugated antibody can confirm tissue-specific deletion

When designing experiments combining knockout models with antibodies, researchers should consider potential compensatory mechanisms that may emerge in knockout systems, as well as the timing of OLFML3 deletion relative to the biological process being studied.

What are common issues encountered when using OLFML3 Antibody, Biotin conjugated, and how can they be resolved?

When working with OLFML3 Antibody, Biotin conjugated, researchers may encounter several technical challenges. Here are methodological solutions to address common issues:

• High background signal in ELISA or immunohistochemistry:

  • Increase blocking time and concentration (try 5% BSA or 10% normal serum from the same species as the secondary detection reagent)

  • Add 0.1-0.3% Triton X-100 to reduce non-specific binding

  • Include avidin/biotin blocking steps to neutralize endogenous biotin

  • Dilute the antibody further (try serial dilutions from 1:500 to 1:5000)

  • Increase washing steps (5-7 washes of 5 minutes each)

• Weak or no signal detection:

  • Verify target expression in your sample (use positive control tissues known to express OLFML3)

  • Check antibody storage conditions (improper storage can lead to degradation)

  • Try antigen retrieval methods for fixed tissues (heat-induced or enzymatic)

  • Increase antibody concentration or incubation time

  • Ensure your detection system (streptavidin-enzyme) is functioning properly

• Cross-reactivity issues:

  • Include additional blocking steps with 5% serum from the same species as your sample

  • Pre-absorb the antibody with proteins from the potentially cross-reactive species

  • Use more stringent washing conditions (higher salt concentration in wash buffer)

  • Validate specificity using OLFML3 knockout controls

• Inconsistent results between experiments:

  • Standardize all protocols, including fixation methods, incubation times, and temperatures

  • Prepare larger batches of diluted antibody and store small aliquots

  • Include reference standards in each experiment

  • Minimize freeze-thaw cycles of the antibody

• Issues with biotin-streptavidin detection:

  • Block endogenous biotin with avidin/biotin blocking kit

  • Ensure streptavidin reagents are fresh and properly stored

  • Consider using amplification systems (tyramide signal amplification) for low abundance targets

By systematically addressing these issues through methodological optimization, researchers can enhance the specificity and sensitivity of experiments using the OLFML3 Antibody, Biotin conjugated.

How should researchers interpret discrepancies between OLFML3 antibody-based detection and mRNA expression data?

Discrepancies between protein detection using OLFML3 antibodies and mRNA expression data are not uncommon and require careful interpretation. Researchers should consider the following methodological approaches:

• Biological explanations for discrepancies:

  • Post-transcriptional regulation: OLFML3 mRNA may be subject to microRNA-mediated suppression or other regulatory mechanisms

  • Protein stability differences: The half-life of OLFML3 protein may differ substantially from its mRNA

  • Secretion dynamics: As a secreted protein , OLFML3 may be produced in one tissue but detected in another

  • Intracellular vs. extracellular localization: Recent research reveals OLFML3 can function both extracellularly and intracellularly , potentially complicating detection

• Technical verification approaches:

  • Validate antibody specificity using recombinant OLFML3 protein and OLFML3 knockout tissues

  • Employ multiple antibodies targeting different OLFML3 epitopes

  • Use alternative protein detection methods:

    • Mass spectrometry-based proteomics

    • Western blotting with size verification

    • Immunoprecipitation followed by mass spectrometry

• Integrated analysis methods:

  • Perform time-course studies to capture potential temporal delays between mRNA expression and protein accumulation

  • Analyze multiple tissue compartments, considering OLFML3's role in different cellular locations

  • Quantify protein at both intracellular and extracellular levels

• Experimental controls:

  • Use siRNA or CRISPR knockout of OLFML3 to verify signal specificity

  • Include correlation analysis with known OLFML3-interacting proteins (e.g., IRG1 )

  • Assess different fixation and permeabilization methods that might affect epitope accessibility

• Documentation and reporting:

  • Clearly report all methodological details to facilitate interpretation

  • Document the specific region of OLFML3 that the antibody targets

  • Consider isoform-specific expression that might explain discrepancies

The literature indicates that OLFML3 has both intracellular and extracellular functions , suggesting that comprehensive analysis should account for both pools of the protein when comparing to mRNA data.

How might OLFML3 Antibody be used to investigate its newly discovered role in mitochondrial function and inflammation?

Recent research has uncovered OLFML3's novel role in promoting IRG1 mitochondrial localization and preventing LPS-induced mitochondrial dysfunction . Researchers can leverage OLFML3 Antibody, Biotin conjugated to further investigate this exciting finding through several methodological approaches:

• Subcellular co-localization studies:

  • Perform immunofluorescence microscopy using the biotin-conjugated OLFML3 antibody (with fluorescent streptavidin) alongside mitochondrial markers

  • Conduct super-resolution microscopy to precisely locate OLFML3 on the outer mitochondrial membrane, as suggested by the literature

  • Quantify co-localization coefficients between OLFML3 and mitochondrial markers under different inflammatory conditions

• Protein interaction network analysis:

  • Use the biotin-conjugated antibody for pull-down experiments followed by mass spectrometry to identify:

    • The complete mitochondrial interactome of OLFML3

    • Changes in interaction partners following LPS or bacterial stimulation

    • Post-translational modifications that might regulate OLFML3's mitochondrial association

• Functional mitochondrial assays:

  • Correlate OLFML3 expression/localization (detected via the antibody) with:

    • Mitochondrial membrane potential measurements

    • Oxygen consumption rate (OCR)

    • Extracellular acidification rate (ECAR)

    • Reactive oxygen species (ROS) production

    • Itaconate levels (the metabolic product of IRG1)

• In vivo inflammation models:

  • Use the antibody for tissue immunohistochemistry in models of:

    • LPS-induced acute lung injury (ALI), where OLFML3 knockout showed exacerbated pathology

    • Bacterial pneumonia models (e.g., Pseudomonas aeruginosa infection)

    • Sepsis models, where OLFML3 knockout reduced survival

• Temporal dynamics studies:

  • Track OLFML3 localization changes over time following inflammatory stimuli

  • Correlate these changes with mitochondrial morphology and function

  • Investigate whether OLFML3 translocation to mitochondria precedes IRG1 localization

The methodological approach should incorporate appropriate controls, including OLFML3 knockout models , to establish specificity and causality in these studies.

What are the emerging therapeutic applications for targeting OLFML3, and how can antibodies facilitate this research?

Emerging research suggests several promising therapeutic applications for targeting OLFML3, with antibodies playing a crucial role in advancing this field:

• Cancer immunotherapy development:

  • The literature indicates that targeting OLFML3 with antibodies inhibits tumor growth in mouse models

  • Researchers can use biotin-conjugated antibodies to:

    • Screen for antibody clones with optimal neutralizing activity

    • Evaluate effects on tumor vascularization and growth

    • Investigate combination therapy with checkpoint inhibitors (anti-PD-1), which showed enhanced efficacy in research models

• Anti-inflammatory therapeutic approaches:

  • Given OLFML3's role in regulating inflammation during acute lung injury and sepsis , antibodies can:

    • Block specific domains of OLFML3 to modulate its interaction with IRG1

    • Target extracellular vs. intracellular pools of OLFML3 selectively

    • Be used to develop biomarkers for patient stratification in inflammatory diseases

• Targeted drug delivery systems:

  • The biotin-conjugated antibody can serve as a targeting moiety for:

    • Nanoparticle-based drug delivery to OLFML3-expressing tissues

    • Antibody-drug conjugates for cancer therapy

    • Imaging agents to visualize OLFML3-expressing tumors or inflammatory sites

• Structure-based drug design:

  • While not directly using the antibody, crystallography studies facilitated by antibody-based purification can:

    • Determine the three-dimensional structure of OLFML3

    • Identify binding pockets for small molecule inhibitors

    • Guide development of domain-specific inhibitors (e.g., targeting the OLF domain that interacts with IRG1 )

• Diagnostic applications:

  • Develop ELISA-based diagnostic tests using the biotin-conjugated antibody to:

    • Measure circulating OLFML3 levels in cancer or inflammatory conditions

    • Correlate OLFML3 levels with disease progression or treatment response

    • Identify patient populations likely to respond to OLFML3-targeted therapies

When pursuing these therapeutic applications, researchers must consider:

• The dual intracellular and extracellular functions of OLFML3

• Potential off-target effects given OLFML3's role in normal physiological processes

• The balance between beneficial anti-tumor effects and potentially detrimental effects on inflammatory responses