IFI27L2 Antibody

What is IFI27L2 and why is it significant in research?

IFI27L2 (Interferon, alpha-Inducible Protein 27-Like 2) is a member of the interferon-stimulated gene (ISG) family with emerging significance in innate immunity and viral infection research . The IFI27 family proteins appear to function as modulators of immune responses, with IFI27 specifically shown to counterbalance innate immune responses during RNA viral infections . Research indicates that IFI27 expression can facilitate viral replication in certain contexts, including influenza virus and SARS-CoV-2, suggesting a complex regulatory role in host-pathogen interactions . Mechanistically, IFI27 achieves this immunomodulatory function through direct RNA binding capabilities and interaction with pattern recognition receptors like RIG-I, making it a critical target for research exploring innate immunity regulation . Understanding IFI27L2's function has implications for viral pathogenesis studies and potential therapeutic approaches targeting excessive inflammatory responses.

What types of IFI27L2 antibodies are available for research applications?

Researchers have access to a diverse range of IFI27L2 antibodies targeting various epitope regions including middle regions (such as AA 81-130) and C-terminal regions of the protein . These antibodies are primarily available as rabbit polyclonal antibodies with varying conjugation options to suit different experimental requirements and detection systems . Conjugate options include unconjugated forms ideal for flexible detection strategies, as well as directly labeled versions with biotin, HRP, or fluorescent tags like FITC and AbBy Fluor® 647 for specialized applications requiring direct detection . Additionally, antibodies are validated for various applications including Western blotting, ELISA, immunohistochemistry (on both frozen and paraffin sections), and various immunofluorescence techniques (cellular and tissue-based) . The species reactivity profile primarily focuses on human samples, with some antibodies also demonstrating reactivity with bovine tissues, allowing for comparative studies across species in some contexts .

How should researchers validate specificity of IFI27L2 antibodies?

Validation of IFI27L2 antibody specificity requires a multi-faceted approach beginning with positive control lysates from cells known to express the target protein, particularly after interferon stimulation or poly(I:C) treatment to upregulate expression . CRISPR/Cas9-generated knockout cell lines provide the gold standard negative control, as demonstrated in studies where IFI27 knockout A549 cells showed complete absence of staining after poly(I:C) treatment, while wild-type cells displayed clear protein expression . Peptide competition assays offer another validation approach, where pre-incubation of the antibody with immunizing peptide should abolish specific signals in subsequent detection applications . Western blot analysis should reveal a band at the expected molecular weight of approximately 11.5 kDa for IFI27 proteins, though post-translational modifications may affect migration patterns . Additionally, cross-reactivity testing against related family members (such as comparing IFI27L2 versus IFI27/ISG12 detection) helps ensure the antibody distinguishes between these structurally similar proteins, particularly important given the incomplete characterization of some family members .

What are the optimal conditions for using IFI27L2 antibodies in Western blotting?

For optimal Western blotting with IFI27L2 antibodies, researchers should begin with careful sample preparation through lysis in RIPA or NP-40 buffer supplemented with protease inhibitors, followed by thorough sonication to ensure complete extraction of this small membrane-associated protein . Protein separation requires special consideration due to IFI27L2's low molecular weight (approximately 11.5 kDa), necessitating higher percentage (15-18%) polyacrylamide gels or specialized gradient gels optimized for small proteins . Transfer conditions should be optimized for small proteins, typically using lower voltage (30-50V) for longer durations (2-3 hours) or semi-dry transfer systems with methanol-containing buffers to enhance retention of small proteins on PVDF membranes (preferred over nitrocellulose for low molecular weight targets) . Blocking should employ 5% non-fat dry milk or BSA in TBS-T for 1-2 hours at room temperature, with primary antibody dilutions typically ranging from 1:500 to 1:2000 depending on the specific antibody preparation, and overnight incubation at 4°C usually yielding optimal results . Positive controls are essential and should include interferon-stimulated cells or tissues known to express IFI27L2, while knockout cell lysates provide excellent negative controls for demonstrating specificity .

How should researchers design immunohistochemistry experiments with IFI27L2 antibodies?

Successful immunohistochemistry (IHC) with IFI27L2 antibodies requires careful attention to tissue fixation and antigen retrieval, with optimal protocols typically involving 10% neutral buffered formalin fixation for 24-48 hours followed by heat-induced epitope retrieval using citrate buffer (pH 6.0) or EDTA buffer (pH 9.0) for 20 minutes . The selection between frozen and paraffin sections should be guided by experimental requirements, with paraffin sections offering superior morphology but potentially requiring more rigorous antigen retrieval, while frozen sections better preserve antigenicity but with compromised tissue architecture . When using biotin-conjugated IFI27L2 antibodies, researchers must implement avidin-biotin blocking steps to minimize endogenous biotin interference, particularly important in tissues with high endogenous biotin like liver, kidney, and brain . Antibody dilutions for IHC applications typically range from 1:50 to 1:200, with optimization recommended through dilution series on known positive control tissues, and incubation periods of 1-2 hours at room temperature or overnight at 4°C typically providing optimal staining . Signal development systems should be matched to the antibody conjugate, with HRP/DAB systems for enzyme-conjugated antibodies and appropriate fluorescence microscopy settings for fluorophore-conjugated versions, always including positive controls (interferon-stimulated tissues) and negative controls (omitting primary antibody and using tissues known to lack expression) .

What controls are essential when using IFI27L2 antibodies in research?

Implementing a comprehensive control strategy for IFI27L2 antibody experiments is critical and should include positive controls using cells or tissues with confirmed IFI27L2 expression, preferably after stimulation with type I interferons or poly(I:C) to ensure detectable expression levels . Negative controls must include technical controls (omitting primary antibody) and biological controls such as CRISPR/Cas9-generated IFI27L2 knockout cells, which have proven invaluable for confirming signal specificity as demonstrated in published knockout validation studies . Isotype controls matched to the primary antibody host species and immunoglobulin class help identify potential non-specific binding, particularly important in flow cytometry and immunofluorescence applications where background fluorescence can confound interpretation . Expression controls using recombinant IFI27L2 protein (tagged or untagged) in overexpression systems provide additional confirmation of antibody specificity and can serve as positive controls across different applications . For comprehensive validation, researchers should also consider cross-reactivity controls testing the antibody against related family members (IFI27, IFI27L1) to ensure target specificity, particularly important given the sequence similarity among these interferon-induced proteins .

How can researchers optimize protocols for fluorescence microscopy with IFI27L2 antibodies?

Optimizing fluorescence microscopy with IFI27L2 antibodies begins with proper sample preparation, including careful fixation preferably with 4% paraformaldehyde for 15-20 minutes to preserve both antigenicity and cellular structures, followed by gentle permeabilization with 0.1-0.3% Triton X-100 for 10 minutes to enable antibody access to intracellular targets . Antibody selection should consider native versus conjugated formats based on experimental needs, with directly conjugated FITC or AbBy Fluor® 647 antibodies simplifying protocols but potentially sacrificing signal amplification, while unconjugated primary antibodies followed by fluorophore-conjugated secondaries offer greater sensitivity and flexibility . Blocking and antibody dilution buffers should contain 1-5% normal serum from the species in which the secondary antibody was raised, with primary antibody incubations typically performed at 1:50-1:200 dilutions overnight at 4°C, and secondary antibody incubations at manufacturer-recommended dilutions (usually 1:200-1:1000) for 1-2 hours at room temperature . To minimize photobleaching, samples should be protected from light during all incubation steps, mounted with anti-fade mounting media containing DAPI for nuclear counterstaining, and imaged promptly or stored at 4°C in the dark . Co-localization studies may be particularly valuable for IFI27L2 research, requiring careful selection of compatible fluorophores with minimal spectral overlap and sequential imaging protocols to avoid bleed-through artifacts .

How can IFI27L2 antibodies be used to study RNA-protein interactions?

Investigation of RNA-protein interactions involving IFI27L2 can be accomplished through immunoprecipitation techniques using IFI27L2 antibodies followed by RNA isolation and analysis, building upon the demonstrated RNA-binding capabilities of the IFI27 family . Researchers should consider RNA immunoprecipitation (RIP) assays where cells are crosslinked with formaldehyde, lysed under non-denaturing conditions, and IFI27L2-RNA complexes are immunoprecipitated using validated antibodies against the middle region or C-terminus of IFI27L2, with subsequent RNA isolation and analysis by RT-qPCR or sequencing . For identifying specific RNA-binding motifs, photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP) protocols can be implemented, requiring metabolic labeling of cells with photoactivatable nucleosides like 4-thiouridine, UV crosslinking, and immunoprecipitation with well-characterized IFI27L2 antibodies like those targeting amino acids 81-130 . In vitro validation of binding interactions can utilize recombinant IFI27L2 protein and synthetic RNA molecules in electrophoretic mobility shift assays (EMSAs) or pull-down assays with biotinylated RNA substrates similar to the poly(I:C) binding studies described in the literature, where IFI27 showed specific binding to poly(I:C) but not poly(C) . These approaches should be complemented with control experiments using IFI27L2 knockout cells generated through CRISPR/Cas9 technology to confirm specificity of observed interactions and rule out antibody cross-reactivity with related family members .

What approaches can be used to study IFI27L2's role in viral infection models?

Studying IFI27L2's role in viral infection requires integrated approaches combining genetic manipulation, antibody-based detection, and functional readouts to fully characterize its impact on viral replication and immune responses . Researchers should develop cellular models with modulated IFI27L2 expression, including CRISPR/Cas9 knockout lines as described in published studies, alongside overexpression systems using tagged or untagged constructs to facilitate detection with either anti-tag or native IFI27L2 antibodies . For infection studies, researcher-selected RNA viruses (particularly influenza and SARS-CoV-2 based on documented interactions) can be used to infect control and IFI27L2-modulated cells, with viral replication monitored through plaque assays, RT-qPCR quantification of viral genomes, or immunofluorescence detection of viral proteins . Downstream analysis should assess innate immune signaling pathways using phospho-specific antibodies for key signaling molecules (IRF3, NFκB), alongside RT-qPCR measurement of interferon-stimulated gene expression and ELISA quantification of secreted cytokines and interferons to establish IFI27L2's immunomodulatory effects . Mechanistic investigations should employ co-immunoprecipitation studies using IFI27L2 antibodies to identify protein-protein interactions, particularly with pattern recognition receptors like RIG-I, followed by functional validation through reporter assays measuring interferon promoter activity in the presence and absence of IFI27L2 .

How can researchers distinguish between IFI27 family members in experimental systems?

Distinguishing between closely related IFI27 family members (IFI27/ISG12, IFI27L1, IFI27L2) requires careful selection of detection reagents and experimental approaches to avoid cross-reactivity and misinterpretation of results . Researchers should prioritize antibodies targeting unique epitopes within IFI27L2 that have minimal sequence homology with other family members, with antibodies against the middle region (AA 81-130) often providing better specificity than those targeting more conserved regions . Validation experiments must include expression controls where each family member is individually overexpressed, followed by Western blot analysis with the selected antibody to confirm detection of only the intended target at the expected molecular weight (approximately 11.5 kDa for IFI27L2) . At the transcript level, RT-qPCR assays should employ primer pairs spanning unique exon junctions specific to IFI27L2, with primer efficiency and specificity validated using synthetic templates or overexpression constructs for each family member . For more complex samples like tissues or primary cells, immunoprecipitation followed by mass spectrometry can provide definitive identification of the captured protein, distinguishing between family members based on unique peptide sequences . Researchers should also consider the differential regulation of family members, as each may respond differently to various stimuli, offering another approach to distinguish their expression patterns across experimental conditions .

What techniques can assess IFI27L2's role in modulating RIG-I-mediated signaling?

To investigate IFI27L2's role in modulating RIG-I-mediated signaling, researchers should implement a multi-faceted approach beginning with co-immunoprecipitation experiments using antibodies against either RIG-I or IFI27L2 to confirm their physical interaction, as suggested by studies of the related IFI27 protein . Functional impact on RIG-I activation can be assessed through RIG-I ATPase activity assays in the presence and absence of recombinant IFI27L2, determining whether IFI27L2 directly inhibits RIG-I's enzymatic activity in response to RNA ligands like poly(I:C) . Researchers should establish cellular models with modified IFI27L2 expression (knockout, knockdown, and overexpression) and measure downstream signaling events following RIG-I stimulation, including IRF3 phosphorylation/nuclear translocation, type I interferon production, and interferon-stimulated gene expression, using appropriate antibodies for detection of these signaling components . RNA competition assays can evaluate whether IFI27L2 competes with RIG-I for RNA binding, utilizing purified components and labeled RNA substrates in binding reactions with varied concentrations of IFI27L2 protein to determine if RIG-I binding is displaced . For in vivo relevance, viral challenge studies in IFI27L2-deficient mice or cells should assess whether the absence of IFI27L2 enhances RIG-I-dependent antiviral responses and restricts viral replication, providing physiological context to the molecular findings .

How should researchers address inconsistent results with IFI27L2 antibodies?

When encountering inconsistent results with IFI27L2 antibodies, researchers should implement a systematic troubleshooting approach beginning with antibody validation through Western blot analysis using positive controls (interferon-stimulated cells) and negative controls (IFI27L2 knockout cells) to confirm specificity under their specific experimental conditions . Expression level variations may underlie inconsistencies, as IFI27L2 is typically expressed at low baseline levels but strongly induced by type I interferons and viral infection, necessitating appropriate stimulation conditions or selection of time points that capture peak expression following induction . Researchers should carefully evaluate sample preparation protocols, as IFI27L2's small size (approximately 11.5 kDa) and potential membrane association may require specialized extraction conditions including proper cell lysis buffers (RIPA or NP-40 with detergents) and sonication to ensure complete solubilization . Antibody storage and handling practices should be reviewed, ensuring antibodies are stored according to manufacturer recommendations (typically aliquoted and kept at -20°C or -80°C) and avoiding repeated freeze-thaw cycles that can degrade antibody quality over time . Technical variables like blocking reagents, antibody dilutions, and incubation conditions should be systematically optimized through controlled experiments to identify optimal parameters for consistent detection across experiments .

What factors affect IFI27L2 expression levels that might impact antibody detection?

IFI27L2 expression exhibits complex regulatory patterns influenced by multiple factors that researchers must consider when interpreting antibody detection results . Type I and type III interferons are primary inducers of IFI27L2 expression, with induction kinetics varying by cell type and specific interferon subtype, requiring researchers to carefully time their experiments to capture peak expression windows, typically 12-24 hours post-stimulation . Viral infection significantly upregulates IFI27L2 expression through both interferon-dependent and possibly interferon-independent mechanisms, with RNA viruses like influenza and SARS-CoV-2 shown to trigger robust expression, suggesting infection models may provide stronger detection signals than uninfected samples . Cell type-specific expression patterns are evident, with epithelial cells and immune cells showing different baseline and induced expression levels, necessitating cell type-appropriate positive controls and potentially different detection protocols optimized for each experimental system . Researchers should also consider post-transcriptional regulation, as mRNA expression levels may not directly correlate with protein abundance due to regulatory mechanisms affecting translation efficiency and protein stability, potentially explaining discrepancies between RT-qPCR and antibody-based detection results . Environmental factors including cell culture conditions, confluence levels, and passage number can further impact expression, requiring standardized protocols to ensure reproducible results across experiments .

How can researchers optimize IFI27L2 antibody performance in different applications?

Optimizing IFI27L2 antibody performance across applications requires application-specific adjustments and careful selection of antibody formats based on experimental requirements . For Western blotting applications, researchers should select unconjugated antibodies validated specifically for this technique, optimize protein loading (typically requiring higher amounts due to potentially low expression levels), and consider enhanced chemiluminescence (ECL) detection systems with extended exposure times to capture low-abundance signals . In immunohistochemistry and immunofluorescence applications, antigen retrieval methods should be systematically compared (citrate buffer pH 6.0 versus EDTA buffer pH 9.0) to identify optimal conditions for epitope exposure, with antibody concentration titrations (typically starting with 1:50-1:200 dilutions) to determine the optimal signal-to-noise ratio for each specific tissue or cell type . For flow cytometry applications, researchers should select directly conjugated antibodies (FITC, AbBy Fluor® 647) to avoid secondary antibody requirements, implement rigorous blocking of Fc receptors in immune cell populations to prevent non-specific binding, and include fluorescence-minus-one (FMO) controls to set accurate gating strategies . ELISA applications benefit from biotin-conjugated antibodies paired with streptavidin-HRP detection systems for signal amplification, requiring careful optimization of coating antibody concentration, sample dilution, and incubation times to maximize sensitivity while maintaining specificity . Cross-application validation provides additional confidence, with researchers encouraged to confirm key findings using multiple detection methods when possible, particularly for novel or controversial observations regarding IFI27L2 function or expression .