ANGPTL5 Antibody

What is ANGPTL5 and what are its primary biological functions?

Angiopoietin-like Protein 5 (ANGPTL5) is a secreted glycoprotein belonging to the angiopoietin-like protein family. While less extensively characterized than some family members, research indicates ANGPTL5 plays roles in:

• Triglyceride metabolism, with rare sequence variations associated with low plasma triglyceride levels

• Potential roles in hematopoietic stem cell (HSC) regulation, particularly through interaction with inhibitory receptors like LILRB2

• May function in signaling pathways involving calcium/calmodulin-dependent protein kinases CAMKII and CAMKIV

Unlike some other ANGPTL family members, ANGPTL5 is not expressed in mice, making it unique among this protein family . Functionally, ANGPTL5 contains a fibrinogen-like domain and can be detected as a secreted protein in specific experimental conditions.

What types of ANGPTL5 antibodies are available for research applications?

Research-grade ANGPTL5 antibodies are available in several formats with distinct properties:

When selecting an antibody, consider your experimental design. Monoclonal antibodies offer consistent reproducibility and specificity for a single epitope, while polyclonal preparations may provide higher sensitivity due to recognition of multiple epitopes. The clone 1B2 monoclonal antibody was raised against full-length recombinant ANGPTL5 protein with GST tag and has confirmed reactivity with human samples .

How should ANGPTL5 antibodies be stored and handled to maintain optimal activity?

Proper storage and handling are critical for maintaining antibody functionality:

For monoclonal antibodies like the Anti-ANGPTL5 Mouse Monoclonal Antibody (clone 1B2):

• Aliquot and store at -20°C or -80°C to avoid repeated freeze-thaw cycles

• Store in PBS (pH 7.4) buffer to maintain stability

• For frequent use, small working aliquots can be stored at 4°C for up to one month

For polyclonal preparations:

• Store at -20°C for long-term storage (up to one year)

• Many are supplied in PBS containing 50% glycerol and 0.02% sodium azide as preservatives

• Avoid repeated freeze-thaw cycles as this can lead to protein denaturation and loss of activity

Researchers should validate antibody performance after extended storage by including appropriate positive and negative controls in their experiments.

What are the recommended protocols for using ANGPTL5 antibodies in Western blot applications?

When utilizing ANGPTL5 antibodies for Western blot analysis, the following methodological guidelines are recommended:

Standard Western Blot Protocol:

• Sample Preparation:

  • For recombinant ANGPTL5: Use purified protein (commercially available as GST-tagged ANGPTL5)

  • For cell/tissue lysates: Prepare using RIPA buffer with protease inhibitors

  • Expected molecular weight: ~44 kDa (calculated) , though glycosylation may affect migration

• Recommended Working Dilutions:

  • For monoclonal antibodies: Begin optimization at 1:1000 dilution

  • For polyclonal antibodies: Use 1:500-1:2000 dilution range

• Detection System:

  • HRP-conjugated secondary antibody specific to host species (anti-mouse for monoclonal, anti-rabbit for polyclonal)

  • Enhanced chemiluminescence (ECL) detection systems are suitable

• Controls:

  • Positive control: Conditioned media from cells expressing recombinant ANGPTL5

  • Negative control: Lysates from cells known not to express ANGPTL5

  • Blocking peptide control: Pre-incubation with immunizing peptide should abolish specific signal

When analyzing expression in human samples, note that ANGPTL5 may be secreted and potentially processed, resulting in multiple bands. Validation using recombinant protein is strongly recommended before experimental applications.

How can researchers validate the specificity of ANGPTL5 antibodies?

Rigorous validation is essential for ensuring antibody specificity and avoiding misleading results:

• Expression System Validation:

  • Transfect cells with ANGPTL5 expression constructs and compare detection in transfected versus non-transfected cells

  • Include constructs with tagged versions (FLAG-tag or GST-tag) that can be detected with independent antibodies

• Knockdown/Knockout Validation:

  • Use RNA interference (shRNA) to suppress ANGPTL5 expression and confirm reduction of signal

  • CRISPR-Cas9 knockout cells provide definitive negative controls

• Peptide Competition Assay:

  • Pre-incubate antibody with excess immunizing peptide to block specific binding sites

  • Signal reduction/elimination confirms epitope specificity

• Multiple Antibody Concordance:

  • Compare detection patterns using antibodies targeting different epitopes

  • Consistency across antibodies increases confidence in specificity

• Mass Spectrometry Validation:

  • For definitive validation, immunoprecipitate the target and confirm identity by mass spectrometry

When testing antibody specificity, researchers should be aware that sequence variations in ANGPTL5 exist, including rare mutations affecting protein secretion . These variations may affect antibody recognition depending on the epitope location.

What experimental conditions should be optimized when performing ELISA with ANGPTL5 antibodies?

ELISA optimization for ANGPTL5 detection requires careful consideration of several parameters:

Key Optimization Parameters:

• Antibody Concentration:

  • Primary antibody: Start with 1:5000-1:20000 dilution for polyclonal antibodies

  • Capture/detection antibody pairings for sandwich ELISA require independent titration

• Sample Preparation:

  • Serum/plasma samples: Consider dilution in blocking buffer (1:10-1:100)

  • Cell culture supernatants: May require concentration for low-abundance detection

  • Recombinant standards: Create standard curves using purified ANGPTL5 (available as GST-tagged)

• Buffer Optimization:

  • Blocking: 1-5% BSA or non-fat dry milk in PBS/TBS

  • Washing: PBS-Tween 0.05-0.1%

  • Sample diluent: May require addition of detergents to reduce background

• Detection System:

  • Colorimetric (TMB substrate) offers broad utility

  • Chemiluminescent systems provide enhanced sensitivity for low-abundance detection

• Incubation Parameters:

  • Coating: Overnight at 4°C for maximum binding

  • Antibody incubations: 1-2 hours at room temperature or overnight at 4°C

When developing ANGPTL5 ELISAs, researchers should consider that ANGPTL family proteins can exist in both monomeric and oligomeric forms, potentially affecting detection. Validation with recombinant protein is essential to establish assay linearity and dynamic range.

How can researchers investigate the interaction between ANGPTL5 and LILRB2 receptor using antibody-based techniques?

The interaction between ANGPTL5 and LILRB2 can be investigated using several antibody-dependent methodologies:

• Co-Immunoprecipitation (Co-IP):

  • Direct method: Immunoprecipitate with anti-LILRB2 antibody and detect ANGPTL5 in precipitates using anti-ANGPTL5 antibodies

  • Reverse approach: Immunoprecipitate ANGPTL5 and detect LILRB2

  • For confirmation of direct interaction, purified proteins can be used as demonstrated in previous research

• Surface Plasmon Resonance (SPR):

  • Immobilize LILRB2-hFc on sensor chip

  • Flow purified ANGPTL5 at varying concentrations

  • Calculate binding kinetics (association/dissociation constants)

  • Previous studies have utilized this technique to validate Angptl-LILRB2 interactions

• Liquid-Phase Binding Assay:

  • Use 125I-labeled GST-ANGPTL5 for quantitative binding studies

  • Determine saturation parameters (previous studies reported half-maximal saturation of 5.5 ± 1.1 nM)

  • Perform competition experiments with unlabeled protein to confirm specificity

• Flow Cytometry-Based Binding Studies:

  • Express LILRB2 on cell surface (stable transfection)

  • Incubate with tagged ANGPTL5 (FLAG-tagged or GST-tagged)

  • Detect binding using antibodies against the tag or directly against ANGPTL5

These techniques can be complemented with functional assays to assess the biological relevance of the interaction, such as examining the effect on hematopoietic stem cell expansion and calcium signaling pathways.

What approaches can be used to study ANGPTL5 variants and their functional effects on protein expression and secretion?

Analysis of ANGPTL5 variants requires integrating molecular and cellular techniques:

• Expression Vector Construction:

  • Generate expression constructs containing wild-type and variant ANGPTL5 sequences

  • Include epitope tags (FLAG, Myc, etc.) for detection independent of conformation

  • Use mammalian expression vectors with strong promoters (CMV) for efficient expression

• Cellular Expression Analysis:

  • Transfect cells (HEK293A cells are commonly used)

  • Assess intracellular expression by Western blot of cell lysates

  • Analyze secretion by examining culture medium for secreted protein

  • Previous studies have identified variants that affect secretion (e.g., several missense mutations prevented ANGPTL5 secretion)

• Pulse-Chase Analysis:

  • Label newly synthesized proteins with radioactive amino acids

  • Chase with non-radioactive medium

  • Immunoprecipitate at various timepoints to track protein fate

  • Can reveal whether variants affect protein stability or secretion kinetics

• Structural Analysis:

  • Computational modeling of mutations using protein structure prediction

  • Correlation of structural changes with observed functional effects

A comparative table of previously studied ANGPTL5 variants illustrates how different mutations affect protein secretion:

This methodological approach allows researchers to classify variants as loss-of-function based on their effects on protein expression and secretion .

How can ANGPTL5 antibodies be utilized in immunohistochemistry studies, and what tissue-specific expression patterns should researchers expect?

While the search results don't specifically address ANGPTL5 immunohistochemistry, researchers can apply standard practices with appropriate modifications:

• Tissue Preparation Considerations:

  • Fixation: 10% neutral buffered formalin is standard

  • Antigen retrieval: Heat-induced epitope retrieval (citrate buffer pH 6.0 or EDTA buffer pH 9.0)

  • Blocking: Use serum from same species as secondary antibody

• Antibody Selection and Optimization:

  • Begin with validated antibodies for Western blot applications

  • Perform dilution series (typically starting at 1:100-1:500)

  • Include positive control tissues (based on known expression patterns)

  • Consider both monoclonal and polyclonal antibodies, as epitope accessibility may differ in fixed tissues

• Expected Expression Patterns:

  • Human ANGPTL5 expression appears to be more restricted than other family members

  • Based on functional studies, expression in hematopoietic tissues is likely

  • Association with triglyceride metabolism suggests potential expression in liver or adipose tissue

• Validation Approaches:

  • RNA expression correlation: Compare protein localization with mRNA expression data

  • Blocking peptide controls: Pre-absorb antibody with immunizing peptide

  • Multiple antibody concordance: Use antibodies against different epitopes

Researchers should note that ANGPTL5 is not expressed in mice , making the selection of positive control tissues important. Human tissues would be required for relevant expression studies.

What are common challenges in detecting ANGPTL5 and how can they be addressed?

Researchers may encounter several technical challenges when working with ANGPTL5:

• Low Expression Levels:

  • Solution: Use more sensitive detection methods (chemiluminescent substrates for Western blot)

  • Consider sample enrichment through immunoprecipitation prior to detection

  • For cell culture experiments, use expression systems with strong promoters

• Secretion and Processing Issues:

  • Challenge: ANGPTL5 is secreted and may undergo post-translational modifications

  • Solution: Analyze both cell lysates and conditioned media

  • Include positive controls of recombinant protein to identify expected molecular weight

• Antibody Cross-Reactivity:

  • Challenge: Potential cross-reactivity with other ANGPTL family members

  • Solution: Validate specificity using recombinant proteins of related family members

  • Consider using monoclonal antibodies for increased specificity

• Protein Aggregation:

  • Challenge: ANGPTL proteins can form oligomers

  • Solution: Include reducing agents in sample buffer

  • Consider non-reducing conditions in parallel to assess oligomeric states

• Variability in Glycosylation:

  • Challenge: Glycosylation patterns may affect antibody recognition

  • Solution: Consider enzymatic deglycosylation (PNGase F treatment) to improve detection consistency

  • Compare migration patterns before and after deglycosylation

Addressing these challenges requires systematic optimization and appropriate controls to ensure reliable and reproducible detection of ANGPTL5.

How should researchers interpret data showing discrepancies between ANGPTL5 protein levels detected by antibodies versus mRNA expression?

Discrepancies between protein and mRNA levels are common in biological systems and can be particularly relevant for secreted proteins like ANGPTL5:

• Post-Transcriptional Regulation Mechanisms:

  • MicroRNA regulation: Investigate whether microRNAs target ANGPTL5 mRNA

  • mRNA stability differences: Measure mRNA half-life using actinomycin D chase experiments

  • Translational efficiency: Analyze polysome association of ANGPTL5 mRNA

• Post-Translational Processing and Secretion:

  • Secretion efficiency: Analyze both intracellular and secreted protein fractions

  • Protein stability: Compare protein half-life to mRNA half-life

  • Known mutations affecting secretion should be considered (as identified in previous studies)

• Technical Considerations:

  • Antibody sensitivity: Validate detection limits with recombinant protein standards

  • Sampling timeframe: Consider temporal shifts between mRNA expression and protein accumulation

  • Sample preparation: Ensure protocols capture the relevant protein pool (membrane-bound, secreted, etc.)

• Biological Interpretation Framework:

  • Tissue-specific regulation: Different tissues may employ distinct regulatory mechanisms

  • Physiological state influence: Consider whether metabolic conditions affect post-transcriptional regulation

  • Compare with other ANGPTL family members as reference points

When encountering such discrepancies, researchers should systematically evaluate each level of regulation rather than assuming technical error.

What strategies can be employed to study the functional interaction between ANGPTL5 and hematopoietic stem cells?

Investigating the functional relationship between ANGPTL5 and hematopoietic stem cells (HSCs) requires specialized methodologies:

• Ex Vivo Expansion Assays:

  • Culture CD34+ hematopoietic stem/progenitor cells with purified ANGPTL5

  • Assess expansion of HSC-enriched populations (CD34+CD38-CD90+ cells)

  • Measure proliferation markers and perform functional colony-forming assays

  • Compare effects of wild-type versus mutant ANGPTL5 proteins

• Signaling Pathway Analysis:

  • Previous research indicates ANGPTL5 treatment induces phosphorylation of CAMKII and CAMKIV

  • Use phospho-specific antibodies in Western blot analysis

  • Employ selective inhibitors to validate pathway dependency

  • Conduct time-course experiments to determine activation kinetics

• Receptor Modulation Studies:

  • LILRB2 receptor has been identified as an ANGPTL5 binding partner

  • Use shRNA to suppress LILRB2 expression and assess effect on ANGPTL5 binding

  • Conduct competition assays with other ANGPTL family members

  • Previous studies showed LILRB2 silencing resulted in decreased repopulation of human cord blood HSCs

• In Vivo Transplantation Assays:

  • Culture human cord blood cells with or without ANGPTL5

  • Transplant into immunodeficient mice (NOD/SCID)

  • Assess human cell repopulation by flow cytometry

  • Previous research demonstrated 17% repopulation from cells cultured with ANGPTL5 versus 1% from LILRB2-knockdown cells

This comprehensive approach integrates molecular, cellular, and in vivo techniques to determine the functional significance of ANGPTL5 in HSC biology.

What emerging techniques might enhance ANGPTL5 antibody applications in research?

Several cutting-edge approaches hold promise for advancing ANGPTL5 research:

• Proximity Ligation Assays (PLA):

  • Allows visualization of protein-protein interactions in situ

  • Could be applied to study ANGPTL5-LILRB2 interactions in native cellular contexts

  • Provides spatial information about interaction sites within cells or tissues

• Super-Resolution Microscopy:

  • Techniques like STORM or PALM provide nanoscale resolution

  • Can resolve subcellular localization of ANGPTL5 beyond conventional microscopy limits

  • Permits co-localization studies with potential interaction partners

• Engineered Antibody Formats:

  • Single-chain variable fragments (scFvs) for improved tissue penetration

  • Bi-specific antibodies to simultaneously target ANGPTL5 and binding partners

  • Nanobodies derived from camelid antibodies for specialized applications

• CRISPR-Cas9 Knock-in Reporter Systems:

  • Generate endogenous fluorescent protein fusions

  • Allows real-time tracking of ANGPTL5 expression and trafficking

  • Avoids overexpression artifacts associated with transfection

• Single-Cell Proteomics:

  • Emerging technologies allow protein quantification at single-cell level

  • Could reveal heterogeneity in ANGPTL5 expression across populations

  • May identify previously unrecognized cell types producing or responding to ANGPTL5

These technological advances could overcome current limitations in studying low-abundance proteins like ANGPTL5 and provide new insights into its biological functions.

How might comparative studies between ANGPTL family members inform our understanding of ANGPTL5 function?

Comparative approaches offer valuable insights into specialized functions of ANGPTL5:

• Evolutionary Analysis:

  • ANGPTL5 is not expressed in mice, suggesting a relatively recent evolutionary origin

  • Comparative genomics across species can identify conserved regulatory elements

  • Phylogenetic analysis of protein domains may reveal functional specialization

• Structure-Function Relationships:

  • Several ANGPTL family members (ANGPTL3, ANGPTL4) inhibit lipoprotein lipase (LPL)

  • ANGPTL5's impact on LPL is less established due to lower expression levels

  • Domain swapping experiments between family members could identify functional regions

• Expression Pattern Comparison:

  • Systematic analysis of tissue-specific expression across family members

  • Identification of shared versus unique regulatory pathways

  • Correlation with physiological processes and disease states

• Receptor Binding Studies:

  • LILRB2 binds multiple ANGPTL family members with different affinities

  • Comparative binding studies can identify structural determinants of receptor specificity

  • Competition assays between family members can reveal binding hierarchies

These comparative approaches can help position ANGPTL5 within the functional landscape of ANGPTL proteins and identify its unique properties versus shared family functions.